Glucose alleviates ammonia-induced inhibition of short-chain fatty acid metabolism in rat colonic epithelial cells

John D. Cremin, Jr., Mark D. Fitch, and Sharon E. Fleming

Department of Nutritional Sciences and Toxicology, University of California, Berkeley, California 94720–3104

Submitted 11 October 2002 ; accepted in final form 7 March 2003


    ABSTRACT
 TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Ammonia decreased metabolism by rat colonic epithelial cells of butyrate and acetate to CO2 and ketones but increased oxidation of glucose and glutamine. Ammonia decreased cellular concentrations of oxaloacetate for all substrates evaluated. The extent to which butyrate carbon was oxidized to CO2 after entering the tricarboxylic acid (TCA) cycle was not significantly influenced by ammonia, suggesting there was no major shift toward efflux of carbon from the TCA cycle. Ammonia reduced entry of butyrate carbon into the TCA cycle, and the proportion of CoA esterified with acetate and butyrate correlated positively with the production of CO2 and ketone bodies. Also, ammonia reduced oxidation of propionate but had no effect on oxidation of 3-hydroxybutyrate. Inclusion of glucose, lactate, or glutamine with butyrate and acetate counteracted the ability of ammonia to decrease their oxidation. In rat colonocytes, it appears that ammonia suppresses short-chain fatty acid (SCFA) oxidation by inhibiting a step before or during their activation. This inhibition is alleviated by glucose and other energy-generating compounds. These results suggest that ammonia may only affect SCFA metabolism in vivo when glucose availability is compromised.

tricarboxylic acid cycle; butyrate; acetate; CO2; ketone bodies; energy.


LEVELS OF AMMONIA ARE NORMALLY much higher in the colonic lumen than in other body fluids. Ammonia concentrations up to 44 mM have been reported in the human colon (28, 37), and concentrations up to 74 mM have been reported in rodents (24, 37). Fecal ammonia concentrations are high also, with mean values of 46 mM reported in one study (2, 30). Ammonia concentrations are greatly affected by intakes of dietary protein, fiber, and carbohydrate, yet it is difficult to accurately predict the effect of diet, perhaps due to interactions between nutrients (24, 26). On the basis of the available data, it appears that the high-protein, high-fat, low-fiber diet typically consumed in America today will result in high concentrations of ammonia in the human colon.

High-protein diets are known to stimulate cellular proliferation of colonic epithelial cells (16) and to stimulate preneoplastic lesions in the liver (42, 43). The stimulatory effect of high dietary protein on cellular proliferation and colonic carcinogenesis has been attributed by some to ammonia (24). The ability of ammonia to induce cellular proliferation, alter DNA synthesis, and alter processes of cell replication, including RNA polymerase activity, has long been recognized (40). Ammonia has been shown to cause significant changes in RNA and protein content of intestinal cells (39), to stimulate proliferation of colonic epithelial cells (19), and to stimulate colonic tumorigenesis (3). The mechanism by which ammonia influences these cellular processes is not known.

The influence of ammonia on cellular metabolism is organ specific. In the brain, increasing ammonia concentrations above the normal low levels of 0.1 mM or less induces coma, limits energy production, at least in part, via inhibition of mitochondrial dehydrogenases (1, 23), and depletes brain ATP by activating Na+/K+-ATPase (11) and by inhibiting the malate-aspartate shuttle (10, 17). By contrast, ammonia was shown to stimulate glycolysis and TCA cycle metabolism of glucose and citrate in chick colonic epithelial cells (33). Although butyrate is a major source of energy for colonic epithelial cells, the interaction between ammonia and butyrate metabolism has not been fully investigated. In the one study that was available, ammonia was reported to inhibit butyrate metabolism (7).

Factors that limit metabolism of butyrate by colonocytes have the potential to negatively impact colonic function, because butyrate provides energy and synthetic precursors to the epithelial cells of the colonic mucosa. A previous observation in chick enterocytes (33) and a preliminary observation in our laboratory (6) that ammonia stimulated glucose oxidation led us to investigate the interaction between these substrates in the presence of ammonia. We hypothesized that butyrate oxidation, if inhibited due to suppression of mitochondrial malate dehydrogenase, would be relieved by glucose. Relief of ammonia-induced suppression of butyrate would alleviate concerns regarding the influence of ammonia on colonic metabolism. Thus, in these studies, we aimed to determine whether the influence of ammonia on glucose and/or short-chain fatty acids (SCFA) would be modulated by substrate interaction and, if so, the basis for this interaction. The results presented here contribute to the general goals of determining the physiological effects of ammonia on colonic epithelial cell energy metabolism and of determining how ammonia exerts its effects. These studies have physiological relevance, because colonic epithelial cells are constantly and uniquely exposed via the lumenal contents to high concentrations of ammonia and butyrate. Inhibiting oxidation of these important compounds by 20% or more could negatively influence availability of energy and synthetic precursors for colonic epithelial cells and could negatively influence cellular processes including regulation of cellular proliferation.


    MATERIALS AND METHODS
 TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Animals. Experiments were conducted using mature (255–380 g, 5–8 mo of age) Fischer 344 male rats (Harlan Industries, Indianapolis, IN). They were allowed free access to NIH 31 stock diet (Western Research Products, Hayward, CA) for at least 7 days before colonic epithelial cells were harvested. All procedures were reviewed and approved by the University of California Berkeley Animal Care and Use Committee.

Chemicals. 14C-labeled compounds were obtained from Dupont New England Nuclear (Boston, MA) and from American Radiolabeled Chemicals (St. Louis, MO). Citrate synthase was obtained from Boehringer-Mannheim (Indianapolis, IN).

14C-labeled glucose, glutamine, and succinate were further purified before use by thin-layer chromatography. The labeled compound was applied to 20 x 20-cm glass or plastic plates coated with MN 300 Cellulose (Brinkman Instruments, Westbury, NY) and developed in a solvent system containing n-butanol:glacial acetic acid:water (24:4:10, vol/vol/vol) as described previously (41). The purified radiolabeled compound was eluted from the cellulose with water that was subsequently removed by lyophilization.

14C-labeled butyrate and acetate were further purified also using thin-layer chromatography as described above. For these compounds, however, the solvent was a two-phase 1:1 (vol/vol) combination of n-butanol (upper phase) to 1.5 M NH4OH (lower phase) (9). The bottom of the glass plate was elevated so that it remained exclusively in the upper phase. Immediately after the plate was developed, it was removed from the tank and lightly sprayed with 0.1 M Na·HEPES (pH 7.4) to prevent volatilization of the radiolabeled compounds from the plate. The radiolabeled compounds were eluted from the cellulose into a small volume of incubation buffer immediately before use.

To control for the effects of increased osmotic pressure and concentration of Cl caused by the addition of NH4Cl to the incubation medium, an equimolar amount of NaCl was substituted for NH4Cl in the control flasks in all experiments. All treatments were duplicated within each replicate.

Colonic epithelial cell isolation and incubation. Colonic epithelial cells were isolated using previously described procedures (15). Briefly, the colon was removed, everted, incubated for 30 min at 37°C in a Ca-free Krebs-Henseleit (KH) buffer containing 2.5 mM dithiothreitol, 5 mM EDTA, and antibiotics [2.5 µg/ml amphotericin, 100 µg/ml kanamycin monosulfate, 250 U/ml penicillin G, and 250 µg/ml streptomycin sulfate; (25)]. The epithelium was subsequently forced from the connective tissue with a pressurized stream of KH buffer containing 5 mM dithiothreitol and 0.025% bovine serum albumin (KHD). Epithelial cells were washed and resuspended in KHD.

Stock solutions of NH4Cl and NaCl were made by dissolving the appropriate amount of compound in KH supplemented with 2.5 mM CaCl2, 0.025% (wt/vol) bovine serum albumin, and antibiotics [0.5 µg/ml amphotericin B, 200 U/ml penicillin G, 200 µg/ml streptomycin sulfate; (15)]. Incubation was initiated by the addition of cell suspension (~2 mg cell dry weight for CO2 experiments; ~6 mg cell dry weight for other experiments) and gassed with 95% O2-5% CO2. Incubations were stopped after 45 min with 0.8 ml methanol and 1.0 ml 1 M NaH2PO4 (for CO2 experiments) or with 0.5 ml of 10% (vol/vol) HClO4.

The viability of the cell suspension prepared for each replicate was monitored by measuring the leakage of lactate dehydrogenase (LDH) into the incubation medium of cells given 5 mM glutamine as a substrate. Leakage of LDH from cells indicates decreased cell membrane integrity. The leakage of LDH from the cells in this control flask (15) averaged 8%, which is comparable to the values observed in our laboratory and in that of others (8).

For ketone body (3-hydroxybutyrate + acetoacetate) and oxaloacetate (OAA) determinations, incubations parallel to the 14CO2 incubations were conducted. Before analyzing for ketone bodies, a 100 µl aliquot of [1-14C]propionic acid solution (10,000 dpm) was added as internal standard. Precipitated protein was removed by centrifugation (2,000 g), and the supernatant was titrated to pH 9–10 with KOH. The resulting KClO4 precipitate was removed by centrifugation (2,000 g), and the supernatant was lyophilized to dryness. To analyze for OAA, the acidified incubation medium was immediately processed and neutralized (31). Samples were stored at –80°C before analysis.

Conversion of substrate carbon to CO2. The specific activities of the substrates used to measure conversion of substrate carbon to CO2 ranged from 20,000 to 115,000 dpm/µmol. Carbon dioxide was collected and 14CO2 quantified (15). Background radioactivity for each treatment was determined in the absence of cell suspension and was subtracted from the corresponding treatment value. Total CO2 production was calculated as previously described (13) and quantified as the rate at which substrate carbon was metabolized to CO2.

As previously discussed (15), the probability that the carbon from exogenous substrates, which enters the TCA cycle, will be oxidized to CO2 vs. other possible metabolic fates was determined by comparing the rates of conversion of trace amounts of very high specific activity [1,4-14C]succinate or [2,3-14C]succinate (at least 1.2 x 108 dpm/µmol, <0.01 mM final concentration of succinate in incubation medium) to 14CO2 in the presence of the exogenous substrates. This probability is equivalent to the A + T value calculated by Mallet et al. (27). The rate of entry of substrate carbon into the TCA cycle was calculated (15) by dividing the rate of CO2 production by the value for A + T.

Conversion of substrate carbon to ketone bodies. Lyophilized salts were extracted four times with 330 µl of 97% ethanol. The combined extracts were titrated to pH 8 using 1% KOH in methanol and universal indicator solution. After evaporation of the ethanol, the dried samples were redissolved in 100 µl water, spiked with 10 mM each of acetate, propionate, butyrate, lactate, and hydroxybutyrate and 0.5 mM acetoacetate, and retitrated to pH 8.

For derivatization before HPLC analysis, the aqueous samples were evaporated to dryness and resuspended in a total volume of 450 µl acetonitrile containing 6.2 nmol 18-crown-6 ether and either 100 µmol 2-bromoacetophenone (for 30 mM acetate substrates) or 25 µmol 2-bromoacetophenone (for 5 mM substrates). Vials were sonicated to disrupt salt crystals and heated at 100°C for 1 h. To obtain complete derivatization, a second redissolution and 2-bromoacetophenone treatment was done by evaporating the acetonitrile, dissolving samples in 100 µl 50% ethanol, evaporating the ethanol-water, resuspending in 350 µl acetonitrile containing an additional 12.5 µmol of 2-bromoacetophenone, and incubating again at 100°C for 30 min.

For HPLC analysis, the samples were evaporated to dryness and resuspended in either 500–600 µl of 60% acetonitrile (for 30 mM acetate substrates) or 500 µl of 50% acetonitrile (for 5 mM substrates). HPLC column and gradient conditions were reported previously (12) and were sufficient to resolve lactate, acetoacetate, hydroxybutyrate, acetate, propionate, and butyrate. Peaks were detected by ultraviolet absorbance, and column effluent was collected and counted in Hionic Fluor LSC cocktail using a 1600-TR liquid scintillation counter (Packard Instruments, Meriden, CT).

Concentration of OAA. Analysis of OAA in neutralized HClO4 extracts was conducted within 24 h of the cell incubation (31). Briefly, the amount of OAA was determined by using citrate synthase to condense exogenous [3H]acetyl-CoA with OAA to form [3H]citrate. Unreacted [3H]acetyl-CoA was removed by activated charcoal and centrifugation. The radioactivity from the [3H]citrate in the supernatant was quantified using liquid scintillation counting. The specific activity of the [3H]acetyl-CoA incubations was 9.95 x 106 dpm/{eta}mol, with a total radioactivity of 300,000 dpm/assay.

The assay for OAA was linear from 0 to 20 {rho}mol of OAA (r = 0.999). Recovery of OAA added to the incubation medium at the end of incubation was linear from 28 to 225 {rho}mol of added OAA and averaged 100.9%. In a preliminary experiment, it was determined that the presence of ammonia did not interfere with the OAA assay.

Net lactate production. Amounts of lactate in neutralized HClO4 extracts were determined enzymatically using an assay kit (Sigma, St. Louis, MO).

CoA derivatives. Acid-soluble acyl-CoA esters were quantified using a modification of the HPLC method of Corkey (4) and Corkey and Deeney (5). Free CoA was protected by adding dithiothreitol (to a final concentration of 1 mM) during TCA precipitation and following ethyl ether extraction. Samples were analyzed using a C18 reverse-phase column (0.46 x 25 cm, Absorbosphere HS 5-µm particles, Alltech, Deerfield, IL) fitted with a guard column. The elution rate (1.0 ml/min) and gradient conditions facilitated separation of free CoA and all major acyl-CoA esters through butyryl-CoA. The mobile phases included 0.1 M KH2PO4 (pH 5.0; buffer A) and 0.1 M KH2PO4 containing 40% acetonitrile (pH 5.0; buffer B). The initial composition of the mobile phase was 5.0% buffer B (0–7 min) and was increased to 10% buffer B over the next 3 min, to 80% buffer B between 20 and 60 min, and to 100% buffer B at 60 min. Absorbance measurements were made at 254 {eta}m. Identification of peaks was based on retention times of standards injected separately and changed together with samples. Disappearance of peaks following alkaline hydrolysis was used also to verify the identity of major CoA compounds. Recovery was similar among the CoA compounds and averaged 90%. Esters were calculated as percentages of the sum of CoA-containing compounds in each sample, because total CoA content did not change during the 45-min period of incubation.

Statistical analyses. Each data set was statistically analyzed by ANOVA. The effects of replicate, ammonia, substrate, and ammonia times substrate interaction were included in the model where appropriate. If the F statistic for an effect was significant, differences among the individual least-squares means for that effect were tested using single degree of freedom contrasts (38). A P value of 0.05 was used as the level of significance for all tests. The analyses were performed using the general linear models procedure of SAS (38). Correlation coefficients and regression analyses were performed using Excel statistical programs.


    RESULTS
 TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Effects of ammonia on oxidative metabolism of substrates to CO2 and ketone bodies. In our initial experiment, the oxidation of butyrate to CO2 was 28% lower for cells incubated in 40 mM NH4Cl vs. equimolar NaCl (Table 1). Acetate oxidation to CO2 was suppressed by 38% when incubated in 40 mM NH4Cl. In contrast, NH4Cl stimulated oxidation to CO2 of glucose by 15% and glutamine oxidation by 10% (Table 2). Net lactate production was significantly increased also (by 10%) by NH4Cl when glucose and butyrate were included in the media (data not shown).


View this table:
[in this window]
[in a new window]
 
Table 1. Influence of NH4Cl on CO2 production from butyrate and acetate

 

View this table:
[in this window]
[in a new window]
 
Table 2. Influence of NH4Cl on CO2 production from glucose and glutamine

 

Adding glucose to the incubation medium counteracted the ability of ammonia to inhibit the oxidation of both butyrate and acetate to CO2 (Table 1). By contrast, adding butyrate to the media did not counteract the stimulatory effect of 40 mM NH4Cl on glucose oxidation to CO2 (Table 2). Also, adding a mixture of substrates (acetate, propionate, butyrate, and glucose) to the media did not counteract the stimulatory effect of 40 mM NH4Cl on the production of CO2 from glutamine.

To determine whether NH4Cl had a general inhibitory effect on SCFA metabolism, the effects of 40 mM NH4Cl on CO2 and ketone body production were simultaneously assessed. In this experiment, NH4Cl suppressed CO2 production from butyrate by 18% and ketone body production from butyrate by 68% (Fig. 1). Glucose largely alleviated this suppression.



View larger version (12K):
[in this window]
[in a new window]
 
Fig. 1. Metabolism of butyrate to both CO2 (A) and ketone bodies (B) is decreased by NH4Cl and largely restored by coincubation with glucose. Columns and bars represent means ± SE, respectively. CO2 and ketone body data were determined in the same experiment (n = 4). Colonocytes were incubated in [1-14C]butyrate, 5 mM butyrate, either 40 mM NaCl or 40 mM NH4Cl, and, where indicated, 5 mM glucose. Means with different superscripts are significantly different at P < 0.05.

 

Influence of substrate and ammonium chloride concentrations on oxidative metabolism. To determine whether NH4Cl suppressed SCFA oxidation only under the conditions unique to our initial experiment, several subsequent experiments were conducted. In one of these experiments, acetate concentrations of both 5 and 30 mM were used, and the production of CO2 and ketone bodies was assessed (Fig. 2). NH4Cl signifi-cantly suppressed the metabolism of acetate to both CO2 and ketone bodies when acetate was present at both low (5 mM) and high (30 mM) concentrations. Because the interaction between substrate concentration and ammonia was not statistically significant, the results indicate that the degree to which ammonia inhibited the oxidation of acetate to CO2 or to ketone bodies was similar for these two concentrations of acetate.



View larger version (14K):
[in this window]
[in a new window]
 
Fig. 2. NH4Cl decreases production of CO2 (A) and ketone bodies (B) from acetate at both 5 and 30 mM. Columns and bars represent means ± SE, respectively. CO2 and ketone body data were attained from a single experiment (n = 4). Colonocytes were incubated in 5 or 30 mM acetate, [U-14C]acetate, and either 40 mM NaCl or 40 mM NH4Cl. Because the interaction between acetate concentration and salts was not statistically significant, differences between NaCl and NH4Cl were determined on the pooled means. Pooled means of the NH4Cl treatments were significantly different from the pooled means of the NaCl treatment (*P < 0.05).

 

In a separate experiment, butyrate concentration was held at 5 mM, whereas NH4Cl was tested at both 10 and 40 mM. CO2 production from butyrate was suppressed to a similar extent by 10 and 40 mM NH4Cl as indicated by the lack of a significant interaction between salts concentration and substrate (Table 3). Glucose alleviated the suppressive effect of NH4Cl on CO2 production from butyrate at both concentrations of salts.


View this table:
[in this window]
[in a new window]
 
Table 3. Influence of salts concentration on CO2 production from butyrate

 

Glucose oxidation to CO2 was assessed at NH4Cl concentrations ranging from 0 to 40 mM. CO2 production from glucose increased with increasing NH4Cl concentrations, approaching a maximal level at the highest concentrations of ammonia tested (Fig. 3).



View larger version (10K):
[in this window]
[in a new window]
 
Fig. 3. The rate of increase in CO2 production from glucose is dependent on NH4Cl concentration. Data points are means ± SE (n = 4), ranged from 0.10–0.26, and were not proportional to the means. Cells were incubated with 20 mM glucose, [U-14C]glucose, and NH4Cl at the concentrations indicated.

 

Effects of ammonia on OAA concentrations and TCA cycle flux. To evaluate our initial hypothesis that glucose would alleviate ammonia-induced suppression of butyrate oxidation to CO2 due to suppression of mitochondrial malate dehydrogenase, we evaluated the influence of ammonia on cellular concentrations of OAA. OAA concentrations were higher when glucose was present either alone or with butyrate than when butyrate was the sole exogenous substrate (Table 4). Also, OAA concentrations were significantly lower following incubation in 40 mM NH4Cl vs. NaCl for each of the substrates tested. Although we hypothesized that suppression of butyrate oxidation by NH4Cl may be due to lack of sufficient OAA to support influx into the TCA cycle, an observation supported by data in brain tissue and mediated via NH4Cl suppression of malate dehydrogenase, changes in OAA concentrations in colonic epithelial cells did not correlate with changes in CO2 production. Whereas NH4Cl suppressed OAA concentrations with all three substrates that were tested, CO2 production was suppressed only when butyrate was the sole exogenous substrate (Table 1, Fig. 1).


View this table:
[in this window]
[in a new window]
 
Table 4. Influence of NH4Cl on cellular concentrations of OAA, A + T, and TCA cycle entry of butyrate

 

Efflux of intermediates from the TCA cycle was hypothesized as a factor that could account for reduced oxidation of butyrate carbon to CO2. Values for "A + T" represent the probability that carbon atoms entering the TCA cycle will be fully oxidized to CO2. Values for A + T would be reduced, for example, if NH4Cl caused increased amounts of {alpha}-ketoglutarate to be converted to glutamine. NH4Cl did not significantly influence values for A + T when butyrate was the sole exogenous substrate (Table 4).

Oxidation of substrates to CO2 could be influenced also by the rates at which substrate carbon enters the TCA cycle. The influence of NH4Cl on butyrate and glutamine carbon entry into the TCA cycle was calculated using substrate oxidation and A + T data. In the presence of NH4Cl, rates of TCA cycle entry were reduced 26% for butyrate, yet increased by ~10% for both glucose and glutamine (Table 4). Thus there appeared to be some relationship between the influence of ammonia on oxidation to CO2 and TCA cycle entry of the substrate.

Influence of ammonia on CoA derivatives. Because NH4Cl suppressed the metabolism of acetate and butyrate to both CO2 and ketone bodies and because ammonia appeared to suppress entry of butyrate carbon into the TCA cycle, we tested the hypothesis that suppression was mediated by reducing availability of free CoA. Because cells were exposed to treatments for only 45 min (insufficient time to influence overall levels of CoA), the percent distribution of a CoA species was assumed to reflect the influence of treatment on their relative concentrations. Malonyl-CoA, acetoacetyl-CoA, hydroxybutyryl-CoA, and hydroxymethyl glutaryl-CoA each accounted for <2% of all CoA species, and treatment did not significantly influence the proportions of these CoA derivatives (data not shown). Also, treatment did not significantly influence the proportion of CoA present as either acetyl-CoA or as butyryl-CoA (Table 5). By contrast, there was a signifi-cant treatment effect on proportion of CoA present as both free CoA and as the sum of acetyl- and butyryl-CoA. Because there was a significant interaction between substrate and salts, differences between treatments were determined among the nine treatments. Compared with NaCl, incubating cells in NH4Cl did not significantly influence the proportion of CoA present as free CoA, acetyl-CoA, butyryl-CoA, or the sum of acetyl- and butyryl-CoA for the substrates tested. Thus the data did not support the hypothesis that ammonia-induced suppression of acetate and butyrate oxidation was mediated by reduced availability of free CoA.


View this table:
[in this window]
[in a new window]
 
Table 5. Influence of NH4Cl on distribution of free CoA and CoA derivatives

 

The relationship between CoA species and SCFA oxidation was considered further by using the data from this experiment to evaluate the correlation between key CoA species and products of SCFA oxidation. A highly significant positive correlation (r = +0.90) was observed between CO2 production and percentage of CoA present as the sum of acetyl- and butyryl-CoA (Fig. 4). The correlation coefficients were further increased (r =–0.95) when oxidation to both CO2 and ketone bodies was considered. Also, a highly significant but negative correlation (r =–0.92) was observed between CO2 production from the 14C-labeled SCFA and percentage of CoA present as free CoA. The correlation coefficients were further increased (r = –0.94) when oxidation to both CO2 and ketone bodies was considered.



View larger version (16K):
[in this window]
[in a new window]
 
Fig. 4. Metabolism of acetate and butyrate to CO2 and ketone bodies is highly correlated to proportion of CoA present as acetyl- and butyryl-CoA and inversely correlated to the proportion of CoA present as free CoA. Data points are means (n = 4). Colonocytes were incubated in 10 mM butyrate or acetate. Correlation coefficients are designated r, and all correlation coefficients were statistically significant at P < 0.05. The data from the 9 treatments shown in Table 5 are included in the regressions of CO2. Ketone body production was assessed for only 5 of these 9 treatments. Thus the regressions of CO2 + ketone bodies included the following 5 treatments: 5 mM acetate* in 10 mM NaCl; 5 mM acetate* in 10 mM NH4Cl; 5 mM butyrate* in 40 mM NaCl; 5 mM butyrate* in 40 mM NH4Cl; and 5 mM butyrate* + 5 mM glucose in 40 mM NH4Cl, where * indicates the 14C-labeled compounds.

 

Influence of ammonia on oxidation of propionate and 3-hydroxybutyrate. Data from the CoA experiment suggested that ammonia exerts its effect, at least in part, by inhibiting the activation of SCFA to acyl-CoA derivatives. To test this possibility, the influence of ammonia on oxidation of propionate and 3-hydroxybutyrate was assessed with the following rationale. Although propionate is activated by the same short-chain acyl CoA synthetase that activates acetate and/or butyrate, the pathway by which propionate is metabolized following activation differs from that of acetate and butyrate. Propionate carbon enters the TCA cycle as succinyl-CoA, whereas acetate and butyrate enter the TCA cycle as acetyl-CoA. If ammonia inhibited SCFA oxidation primarily at the point of activation rather than via TCA cycle metabolism downstream of activation, we hypothesized that ammonia would suppress propionate oxidation to an extent similar to that observed for other SCFAs. As observed with acetate and butyrate, propionate oxidation to CO2 was significantly suppressed in the presence of ammonium chloride, and this suppression was largely alleviated by glucose (Fig. 5) as we observed previously for the other SCFA.



View larger version (14K):
[in this window]
[in a new window]
 
Fig. 5. NH4Cl decreases production of CO2 from propionate (A) but not from 3-hydroxybutyrate (B) and glucose (Glc) reverses the inhibition. Columns and bars represent means ± SE, respectively. Colonocytes were incubated in 5 mM propionate and [1-14C]propionate or in 5 mM 3-hydroxybutyrate and [1-14C]3-hydroxybutyrate. Where indicated, cells were incubated in 10 mM NaCl, 10 mM NH4Cl, or 10 mM NH4Cl plus 5 mM glucose. Within panels, means with different lower case letters are significantly different at P < 0.05.

 

Our hypothesis that ammonia reduces SCFA oxidation by inhibiting activation was tested further by evaluating the influence of ammonia on oxidation of 3-hydroxybutyrate to CO2. During metabolism, 3-hydroxybutyrate isomerizes to acetoacetate, which is activated in the mitochondria via the energy-neutral transfer of CoA from succinyl-CoA, and the acetoacetyl-CoA molecules subsequently enter the TCA cycle as acetyl-CoA units. Thus, except for the process of activation, identical pathways should be used during the oxidation of acetate, butyrate, and 3-hydroxybutyrate-derived acetyl-CoA units to CO2. Our results indicate that oxidation of 3-hydroxybutyrate to CO2 is not significantly influenced by ammonia (Fig. 5), supporting the hypothesis that ammonia suppresses an energy-requiring step before or during activation of SCFA.

Ability of substrates other than glucose to alleviate the effects of ammonia on SCFA oxidation. To further evaluate the properties of glucose responsible for alleviating the inhibitory effect of ammonia on oxidation of SCFAs, the effects of other compounds were evaluated. To determine the influence of other pyruvate-generating compounds, lactate and alanine were evaluated. To determine the influence primarily of energy, glutamine was evaluated. As observed with glucose, lactate and glutamine were able to alleviate ammonia-induced suppression of butyrate to CO2 (Fig. 6). By contrast, alanine caused further suppression of butyrate oxidation in the presence of NH4Cl. In parallel experiments, CO2 production from lactate in the presence of butyrate was stimulated by ammonia (4.09 and 3.28 µmol CO2/g x min, 10 mM NH4Cl and NaCl, respectively), and earlier experiments demonstrated that glucose and glutamine were metabolized also to CO2 (Table 2). By contrast, CO2 production from alanine in the presence of butyrate was low and not stimulated by ammonia (0.62 and 1.13 µmol CO2/g x min, 10 mM NH4Cl and NaCl, respectively). Thus the inhibition of SCFA metabolism by ammonia was alleviated only by substrates able to yield energy when metabolized by colonocytes.



View larger version (13K):
[in this window]
[in a new window]
 
Fig. 6. The inhibitory effect of NH4Cl on CO2 production from butyrate is alleviated by glucose, lactate, and glutamine, but not by alanine. Columns and bars represent means ± SE, respectively. Colonocytes were incubated in 5 mM butyrate, [1-14C]butyrate, and either 10 mM NaCl or 10 mM NH4Cl. Where indicated, cells were incubated also in the presence of 5 mM glucose, lactate, glutamine, or alanine. Means with different lower case letters are significantly different at P < 0.05.

 


    DISCUSSION
 TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Ammonia inhibits the oxidation of exogenous butyrate and acetate by colonic epithelial cells. The results of these studies show that ammonia inhibited the oxidation of both butyrate and acetate carbon to CO2 and ketone bodies. In these experiments, ammonia suppressed oxidation of acetate and butyrate to CO2 by 20–40% and to ketone bodies by 50–70%. The magnitude of these changes could have marked effects on the availability of energy and synthetic precursors needed for colonocytes to perform essential functions and maintain their high rates of proliferation.

The observation that ammonia inhibited the oxidation of butyrate carbon to CO2 and ketone bodies corroborates previous observations using colonic epithelial cells isolated from pigs (7). Suppression by ammonia of both CO2 and ketone body production from both acetate and butyrate suggests that ammonia exerts its effect via one or more elements shared by the oxidative pathways leading to CO2 and ketone bodies and shared by acetate and butyrate. We sought to investigate explanations for these observations.

Ammonia stimulates the oxidation of exogenous glucose, glutamine and lactate. Our observation that ammonia increases the oxidation of glucose to CO2 supports a previous observation using chick enterocytes (33). Differences between species or between gut segments may explain why ammonia did not stimulate glucose oxidation to CO2 in pig colonocytes (7). Our results suggest that ammonia increased glucose oxidation in rat colonocytes, at least in part, by reducing efflux of carbon from the TCA cycle and increasing the efficiency of oxidation. This effect is unlikely to fully account for the increased oxidation of glucose to CO2, because glucose carbon entry into the TCA cycle was increased also. The influence of ammonia on energy production from glucose is evident from the increase in CO2 production. Also, ammonia increased net lactate production in the presence of glucose, indicating that the influence of ammonia on energy production from glucose is in excess of the change in CO2 production. Ammonia has been shown to stimulate glycolysis in chick enterocytes (33), and in other tissues, ammonia has been shown to specifically stimulate several enzymes of the glycolytic pathway (29, 36). Our own data showed that ammonia stimulated production of lactate when glucose was present in the media. Together, these data suggest that ammonia increased both glycolysis and oxidative metabolism of glucose in rat colonocytes.

Our results indicate that ammonia stimulates oxidation of substrates other than glucose. Here, we show that ammonia stimulated the oxidation of both glutamine and lactate to CO2. The increased oxidation of glutamine to CO2 appears not to be due to changes in the efficiency with which glutamine was oxidized by the TCA cycle, but rather due to increased entry of glutamine carbon into the TCA cycle following ammonia treatment. The method used in these experiments to measure TCA cycle entry has been shown to provide estimates that are proportional to values derived using [1-14C]glutamine to measure glutamine entry (21, 34).

Ammonia may inhibit SCFA oxidation by suppressing a step before or during their activation. On the basis of observations in other biological tissues (17), we initially tested whether ammonia reduced concentrations of OAA. Others have shown that ammonia suppressed the TCA cycle enzyme malate dehydrogenase, reducing mitochondrial OAA concentrations and limiting entry of acetyl-CoA into the TCA cycle. This had the potential to explain inhibition by ammonia of CO2 production from SCFA and inhibition of SCFA carbon entry into the TCA cycle. Consistent with this hypothesis, addition of glucose to butyrate incubated in the presence of ammonia resulted in elevated cellular OAA concentrations. There was no consistent relationship, however, between CO2 production and OAA concentration across the range of substrates evaluated in this study. Also, ammonia failed to inhibit oxidation of 3-hydroxybutyrate to CO2. This pathway involves conversion of 3-hydroxybutyrate to acetyl-CoA which, subsequently, is oxidized exclusively in the TCA cycle. This observation suggests that ammonia does not inhibit SCFA oxidation solely at the level of the TCA cycle. In support of this, ammonia suppressed the oxidation of SCFA to both CO2 and ketone bodies. Ketone bodies can be produced from butyrate via pathways that do not require entry into the TCA cycle. Thus we reasoned that any decrease in SCFA carbon entry into the TCA cycle would be the result of some earlier ammonia-inhibited event. Because the TCA cycle is unlikely to be involved in ketone body production from SCFA, we reasoned also that changes in the values for A + T also would be the result of an ammonia-inhibited event preceding TCA cycle entry. Consequently, we sought an explanation for inhibition of SCFA oxidation by ammonia in processes upstream of acetyl-CoA and the TCA cycle.

Evidence that activation is directly influenced by ammonia is provided by our observation that ammonia suppressed propionate and butyrate oxidation to CO2 to similar extents. As propionate is metabolized by a pathway distinct from that of acetate and butyrate, these results point also to activation as one of the few steps common to the three SCFA that were evaluated. As evidence that ammonia exerts its effects on SCFA oxidation via elements upstream of acetyl-CoA, we observed that ammonia did not suppress oxidation of 3-hydroxybutyrate to CO2. Ketone bodies may be activated in the mitochondria via a CoA transferase enzyme that is distinct from acyl-CoA synthetase enzymes and via a reaction that does not require ATP. That ammonia might exert its effect via an energy-requiring transport step needed for the oxidation of SCFA, but not ketone bodies, cannot be ruled out.

Glucose alleviates the inhibition of SCFA oxidation by ammonia. Inclusion of glucose counteracted the ability of ammonia to decrease oxidation of acetate, propionate, and butyrate. This is an important observation because it suggests that glucose derived either arterially or lumenally could moderate the inhibition of ammonia on butyrate and acetate oxidation. This has important consequences in vivo, because colonic epithelial cells are constantly exposed to high concentrations of lumenal ammonia, butyrate, and acetate.

Glucose was not unique in its ability to alleviate inhibition of SCFA oxidation by ammonia. Both lactate and glutamine alleviated the inhibition of ammonia, whereas alanine did not. Interestingly, alanine was metabolized to CO2 to a lesser extent than were glucose, glutamine, and lactate. Also, ammonia stimulated oxidation to CO2 of glucose, glutamine, and lactate, whereas alanine oxidation was not significantly affected. These observations suggest that glucose may alleviate the inhibition by contributing energy needed at some step before the formation of an activated SCFA species. Energy-requiring steps include the activation of SCFA via acyl-CoA synthetase enzymes and energy for the transport of substrates and essential cofactors. It seems unlikely that pH is involved, because it is not known how glucose, lactate, and glutamine would equally influence either the cytosolic or mitochondrial pH in a way that inhibition of transport or activation would be alleviated. A potential molecular mechanism by which ammonia could increase the demand for cellular energy is by altering the efficiency of ion flux across the cellular membrane, because ammonia alters intracellular pH (22, 35) and Cl and K+ transport (18, 32) by colonic epithelial cells. Studies that measure directly the influence of ammonia on mitochondrial ATP concentrations were outside the scope of the current studies.

The current results were obtained using isolated colonic epithelial cells. Although it is not known whether ammonia would have the same effects in vivo, our previous results indicate important parallels in the metabolism of SCFA by isolated colonocytes and the colonic mucosa in vivo (1214, 20). Performing these experiments in vivo would provide a unique challenge, because it would be difficult to test the influence of glucose at concentrations below that of the arterial blood. Glucose (and oxygen) delivery to the colonic mucosa is diminished in the presence of vascular disease or when the viscosity of the blood is increased. These changes may occur with diseases including diabetes, chronic radiation injury, and sickle cell disease; with the taking of oral contraceptives, diuretics, and estrogens, etc; with mass lesions as seen with carcinoma and diverticular disease; and with obstruction, fecal impaction, etc. Although beyond the scope of the current studies, models that simulate the influence of compromised vascular perfusion might yield particularly interesting results. The current findings using isolated colonocytes suggest that ammonia will significantly suppress metabolism of acetate and butyrate when glucose availability is reduced due to diminished vascular perfusion.


    ACKNOWLEDGMENTS
 
We thank R. Gill and C. Garza-Feramisco for technical assistance.

This research was funded by the Agriculture Experiment Station and a competitive grant from the National Institute on Aging AG-10765.


    FOOTNOTES
 

Address for reprint requests and other correspondence: S. E. Fleming, 119 Morgan Hall, Dept. of Nutritional Sciences and Toxicology, Univ. of California, Berkeley, CA 94720–3104 (E-mail: fleming{at}nature.berkeley.edu).

The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.


    REFERENCES
 TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 

  1. Butterworth RF. Effects of hyperammonaemia on brain function. J Inherit Metab Dis 21: 6–20, 1998.[ISI][Medline]
  2. Clausen MR and Mortensen PB. Fecal ammonia in patients with adenomatous polyps and cancer of the colon. Nutr Cancer 18: 175–180, 1992.[ISI][Medline]
  3. Clinton SK, Bostwick DG, Olson LM, Mangian HJ, and Visek WJ. Effects of ammonium acetate and sodium cholate on N-methyl-N'-nitro-N-nitrosoguanidine-induced colon carcinogenesis of rats. Cancer Res 48: 3035–3039, 1988.[Abstract]
  4. Corkey BE. Analysis of acyl-coenzyme A esters in biological samples. Methods Enzymol 166: 55–70, 1988.[ISI][Medline]
  5. Corkey BE and Deeney JT. Acyl CoA regulation of metabolism and signal transduction. Prog Clin Biol Res 321: 217–232, 1990.[Medline]
  6. Cremin JD Jr and Fleming SE. Ammonia affects metabolism of glucose, glutamine and butyrate by isolated rat colonic epithelial cells (Abstract). FASEB J 9: A306, 1995.
  7. Darcy-Vrillon B, Cherbuy C, Morel MT, Durand M, and Duee PH. Short chain fatty acid and glucose metabolism in isolated pig colonocytes: modulation by . Mol Cell Biochem 156: 145–151, 1996.[ISI][Medline]
  8. Del Castillo JR, Ricabarra B, and Sulbaran-Carrasco MC. Intermediary metabolism and its relationship with ion transport in isolated guinea pig colonic epithelial cells. Am J Physiol Cell Physiol 260: C626–C634, 1991.[Abstract/Free Full Text]
  9. Duncan REB and Porteaus JW. Identification and determination of the lower straight-chain fatty acids. Analyst 78: 641–646, 1953.[ISI]
  10. Faff-Michalak L and Albrecht J. Aspartate aminotransferase, malate dehydrogenase, and pyruvate carboxylase activities in rat cerebral synaptic and nonsynaptic mitochondria: effects of in vitro treatment with ammonia, hyperammonemia and hepatic encephalopathy. Metab Brain Dis 6: 187–197, 1991.[ISI][Medline]
  11. Felipo V, Kosenko E, Minana MD, Marcaida G, and Grisolia S. Molecular mechanism of acute ammonia toxicity and of its prevention by L-carnitine. Adv Exp Med Biol 368: 65–77, 1994.[Medline]
  12. Fitch MD and Fleming SE. Metabolism of short-chain fatty acids by rat colonic mucosa in vivo. Am J Physiol Gastrointest Liver Physiol 277: G31–G40, 1999.[Abstract/Free Full Text]
  13. Fleming SE, Fitch MD, DeVries S, Liu ML, and Kight C. Nutrient utilization by cells isolated from rat jejunum, cecum and colon. J Nutr 121: 869–878, 1991.[ISI][Medline]
  14. Fleming SE and Gill R. Aging stimulates fatty acid oxidation in rat colonocytes but does not influence the response to dietary fiber. J Gerontol A Biol Sci Med Sci 52: B318–B330, 1997.[Abstract]
  15. Fleming SE and Kight CE. The TCA cycle as an oxidative and synthetic pathway is suppressed with aging in jejunal epithelial cells. Can J Physiol Pharmacol 72: 266–274, 1994.[ISI][Medline]
  16. Fleming SE, Youngman LD, and Ames BN. Intestinal cell proliferation is influenced by intakes of protein and energy, aflatoxin, and whole-body radiation. Nutr Cancer 22: 11–30, 1994.[ISI][Medline]
  17. Hindfelt B, Plum F, and Duffy TE. Effect of acute ammonia intoxication on cerebral metabolism in rats with portacaval shunts. J Clin Invest 59: 386–396, 1977.[ISI][Medline]
  18. Hrnjez BJ, Song JC, Prasad M, Mayol JM, and Matthews JB. Ammonia blockade of intestinal epithelial K+ conductance. Am J Physiol Gastrointest Liver Physiol 277: G521–G532, 1999.[Abstract/Free Full Text]
  19. Ichikawa H and Sakata T. Stimulation of epithelial cell proliferation of isolated distal colon of rats by continuous colonic infusion of ammonia or short-chain fatty acids is nonadditive. J Nutr 128: 843–847, 1998.[Abstract/Free Full Text]
  20. Jorgensen JR, Fitch MD, Mortensen PB, and Fleming SE. In vivo absorption of medium-chain fatty acids by the rat colon exceeds that of short-chain fatty acids. Gastroenterology 120: 1152–1161, 2001.[ISI][Medline]
  21. Kight CE and Fleming SE. Nutrient oxidation by rat intestinal epithelial cells is concentration dependent. J Nutr 123: 876–882, 1993.[ISI][Medline]
  22. King GG, Lohrmann WE, Ickes JW Jr, and Feldman GM. Identification of Na+/H+ exchange on the apical side of surface colonocytes using BCECF. Am J Physiol Gastrointest Liver Physiol 267: G119–G128, 1994.[Abstract/Free Full Text]
  23. Lai JC and Cooper AJ. Neurotoxicity of ammonia and fatty acids: differential inhibition of mitochondrial dehydrogenases by ammonia and fatty acyl coenzyme A derivatives. Neurochem Res 16: 795–803, 1991.[ISI][Medline]
  24. Lin HC and Visek WJ. Large intestinal pH and ammonia in rats: dietary fat and protein interactions. J Nutr 121: 832–843, 1991.[ISI][Medline]
  25. Lundqvist C, Hammarstrom ML, Athlin L, and Hammarstrom S. Isolation of functionally active intraepithelial lymphocytes and enterocytes from human small and large intestine. J Immunol Methods 152: 253–263, 1992.[ISI][Medline]
  26. Lupton JR and Marchant LJ. Independent effects of fiber and protein on colonic luminal ammonia concentration. J Nutr 119: 235–241, 1989.[ISI][Medline]
  27. Mallet RT, Kelleher JK, and Jackson MJ. Substrate metabolism of isolated jejunal epithelium: conservation of three-carbon units. Am J Physiol Cell Physiol 250: C191–C198, 1986.[Abstract/Free Full Text]
  28. Metcalfe-Gibson A, Ing TS, Kuiper JJ, Richards P, Ward EE, and Wrong OM. In vivo dialysis of faeces as a method of stool analysis. II. The influence of diet. Clin Sci (Lond) 33: 89–100, 1967.[ISI][Medline]
  29. Muntz JA and Hurwitz J. The effect of ammonium ions upon isolated reactions of the glycolytic scheme. Arch Biochem Biophys 32: 137–149, 1951.[ISI]
  30. Nordgaard-Andersen I, Clausen MR, and Mortensen PB. Short-chain fatty acids, lactate, and ammonia in ileorectal and ileal pouch contents: a model of cecal fermentation. J Parent Ent Nutr 34: 324–331, 1993.
  31. Pande SV. Oxaloacetate: radiometric method. In: Methods of Enzymatic Analysis, edited by Bergemeyer HU. Weinheim, Germany: Verlag Chemie, 1985, p. 71–79.
  32. Prasad M, Smith JA, Resnick A, Awtrey CS, Hrnjez BJ, and Matthews JB. Ammonia inhibits cAMP-regulated intestinal Cl transport. Asymmetric effects of apical and basolateral exposure and implications for epithelial barrier function. J Clin Invest 96: 2142–2151, 1995.[ISI][Medline]
  33. Prior RL, Topping DC, and Visek WJ. Metabolism of isolated chick small intestinal cells. Effects of ammonia and various salts. Biochemistry 13: 178–183, 1974.[ISI][Medline]
  34. Quan J, Fitch MD, and Fleming SE. Rate at which glutamine enters TCA cycle influences carbon atom fate in intestinal epithelial cells. Am J Physiol Gastrointest Liver Physiol 275: G1299–G1308, 1998.[Abstract/Free Full Text]
  35. Ramirez M, Fernandez R, and Malnic G. Permeation of and cell pH in colonic crypts of the rat. Pflügers Arch 438: 508–515, 1999.[ISI][Medline]
  36. Ratnakumari L and Murthy CR. Response of rat cerebral glycolytic enzymes to hyperammonemic states. Neurosci Lett 161: 37–40, 1993.[ISI][Medline]
  37. Remesy C and Demigne C. Specific effects of fermentable carbohydrates on blood urea flux and ammonia absorption in the rat cecum. J Nutr 119: 560–565, 1989.[ISI][Medline]
  38. SAS. SAS/STAT User's Guide, Version 6 (4th ed.) Cary, NC: SAS Institute, 1989.
  39. Topping DC and Visek WJ. Synthesis of macromolecules by intestinal cells incubated with ammonia. Am J Physiol Endocrinol Gastrointest Metab 233: E341–E347, 1977.
  40. Visek WJ. Diet and cell growth modulation by ammonia. Am J Clin Nutr 31 Suppl 10: S216–S220, 1978.[Abstract]
  41. Windmueller HG and Spaeth AE. Uptake and metabolism of plasma glutamine by the small intestine. J Biol Chem 249: 5070–5079, 1974.[Abstract/Free Full Text]
  42. Youngman LD and Campbell TC. High protein intake promotes the growth of hepatic preneoplastic foci in Fischer 344 rats: evidence that early remodeled foci retain the potential for future growth. J Nutr 121: 1454–1461, 1991.[ISI][Medline]
  43. Youngman LD and Campbell TC. The sustained development of preneoplastic lesions depends on high protein intake. Nutr Cancer 18: 131–142, 1992.[ISI][Medline]