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Nutrient Metabolism

Acetate and Butyrate Are the Major Substrates for De Novo Lipogenesis in Rat Colonic Epithelial Cells^1,2

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

³To whom correspondence should be addressed. E-mail: fleming{at}nature.berkeley.edu.

	ABSTRACT

TOP ABSTRACT MATERIALS AND METHODS RESULTS DISCUSSION LITERATURE CITED

 
The objective of these experiments was to investigate the source of substrates used for lipid synthesis and the pathways of substrate incorporation into lipids by epithelial cells of the colon. Within replicates, cells were exposed to all treatments evaluated in that experiment. By comparing the relative incorporation rates of several ¹⁴C-labeled substrates into lipids, acetate made a significantly larger carbon contribution to lipids than propionate, butyrate, glucose or glutamine under the in vitro conditions utilized in this study. Other major carbon contributors were butyrate and 3-hydroxybutyrate. Glucose, glutamine and propionate made only minor contributions. (-)-Hydroxycitrate did not affect the incorporation of acetate or butyrate carbon into lipids, even though it inhibited colonic ATP-citrate lyase. These data suggest that SCFA carbon used in the synthesis of lipids by colonocytes is not likely transported to the cytosol as citrate. Competition experiments suggest that ketone bodies and butyrate contribute to a single precursor pool for lipogenesis. Ketone bodies did not significantly suppress acetate incorporation into lipid, however. Incorporation of ³H₂O and ¹⁴C-acetate was significantly greater into phospholipids than into free fatty acids and triacylglycerides, suggesting that the major role of lipogenesis is for membrane synthesis. In conclusion, colonocytes appear to synthesize lipids using a pathway distinct from the liver by incorporating mainly SCFA and ketone bodies into lipids, and by using citrate to a limited extent, if at all, to transport acetyl units from the mitochondria to the cytosol.

KEY WORDS: • cholesterol • sterols • fatty acids

The epithelial cells of the colonic mucosa function as a barrier between luminal bacteria and the rest of the body, and as transporters of certain ions, water and SCFA. The maintenance of barrier and the transporter functions depend on the proper assembly of cell membranes, which require cholesterol and phospholipid. Like other rapidly dividing cells (e.g., lymphocytes, thymocytes or tumor cells), colonic mucosal cells have a greater need for cholesterol and phospholipid than slowly dividing cells. This need for lipids can be met by de novo synthesis or by removal of cholesterol and fatty acids from the bloodstream. Although colonic epithelial cells possess LDL receptors, endogenous synthesis supplies the majority of the cellular cholesterol needs, and the uptake of LDL cholesterol is quantitatively less important and essentially constant (1). Although butyrate has been shown to provide some of the acetyl-CoA (AcCoA)³ needed for de novo lipogenesis, it was not known whether butyrate was essential or the preferred substrate for this function.

Factors that affect synthesis of cholesterol and fatty acids have been shown to influence proliferation of colonic cells and risk of colonic tumorigenesis. Using cultured human adenocarcinoma cells, cell proliferation was reduced by blocking the cholesterol synthetic pathway using inhibitors of the regulatory enzyme, hydroxymethyl glutaryl (HMG)-CoA reductase (2–4). In mice, inhibitors of HMG-CoA reductase reduced colonic tumorigenesis (5), and this reduction appeared to be related to suppression of colonic mucosal cholesterol rather than plasma cholesterol (6,7). Conversely, feeding dietary cholesterol increased cell proliferation and risk of colonic tumorigenesis (8,9).

Very little is known about de novo lipogenesis in colonic epithelial cells even though it has been known for some time that colonic tissue is metabolically capable of synthesizing lipids (10,11). Roediger (12) was the first investigator to provide direct evidence for cholesterol and fatty acid synthesis in the epithelial cells of the colon. Carbon atoms from butyrate are incorporated into lipids of rat colonocytes (12) and HT29 cultured human adenocarcinoma cells (13). In these earlier studies, glucose was shown to stimulate lipid synthesis (12,13). Butyrate was shown also to influence lipid metabolism in Caco2 cells (14,15). Whether butyrate is the primary source of synthetic precursors for fatty acid and cholesterol synthesis by nontransformed colonic epithelial cells is unknown.

The primary goal of the present study was to determine which substrates contribute carbon atoms for the synthesis of fatty acids and cholesterol by primary colonic epithelial cells, and the relative contribution of major substrates to the synthetic precursor pools. In this study, substrates including the SCFA, acetate, propionate and butyrate, along with glucose, glutamine and ketone bodies were studied. Colonocytes preferentially metabolize lumenal compared with serosal substrates (16–18). The SCFA produced by the bacterial fermentation of undigested carbohydrate are the major lumenal substrates available to the colonocytes. Butyrate is utilized to a greater extent than acetate and propionate and is the preferred oxidative fuel of the colonic epithelial cell (17–19). If the metabolic pathways and pools used to synthesize lipids were the same as those used to produce CO₂ and ketone bodies, we would expect butyrate to make the greatest carbon contribution to cholesterol and fatty acids in isolated colonocytes. Because butyrate oxidation to CO₂ was not suppressed by the presence of alternative fuels in our earlier studies, we would expect also that butyrate incorporation into lipids would not be influenced by the presence of other substrates. The results of our initial lipogenesis experiments suggested otherwise. Thus, the second goal of the current studies was to attain insight into the pathways by which SCFA are metabolized and transported into the precursor pools for lipid synthesis in colonic epithelial cells.

	MATERIALS AND METHODS

TOP ABSTRACT MATERIALS AND METHODS RESULTS DISCUSSION LITERATURE CITED

 
Animals.

Male Fisher 344 rats (National Institute on Aging breeding colony, Harlan Industries, Indianapolis, IN) weighing 240–400 g were used. Rats were fed a purchased (Western Research Products, Hayward, CA) NIH 31 stock diet (20). All rats had free access to food and water. Rats were housed at 25°C with a 12-h light:dark cycle. The Animal Care and Use Committee, University of California, Berkeley, approved all rat handling procedures.

Chemicals.

Radiochemicals were obtained commercially and included [2-¹⁴C]propionate, [U-¹⁴C]glutamine, [1-¹⁴C]-3-hydroxybutyrate, [1,5-¹⁴C]citrate, [³H]H₂O (American Radiolabeled Chemicals, St. Louis, MO); [2-¹⁴C]acetate, [U-¹⁴C]glucose, [4-¹⁴C]cholesterol (DuPont NEN, Boston, MA); [U-¹⁴C]acetate, [1-¹⁴C]palmitate, [1-¹⁴C]butyrate (ARC; DuPont) and [1-¹⁴C]acetate (ARC; ICN Pharmaceuticals, Costa Mesa, CA). Before use, radioactive tracers were chromatographed via TLC and purified. (-)-Hydroxycitrate (lactone form) was a generous gift from Dr. J. Sepinwall at Hoffmann-LaRoche, Nutley, NJ. The lactone was hydrolyzed to the acid form (21), and the presence of the acid form was confirmed by TLC (22). All other chemicals and reagents were obtained commercially and were reagent grade.

Preparation of isolated cells.

On the day of the experiment, rats were anesthetized by an intraperitoneal injection of Nembutal (Abbott Laboratories, North Chicago, IL) at 5 mg/100 g body weight and killed by heart puncture. The entire colon was removed from the ceco-colonic junction to the distal anal canal. The method for removing the epithelial cells was described previously (23). Lactate dehydrogenase (LDH; EC 1.1.1.27) release was used to evaluate membrane integrity (23). Leakage of LDH into the medium was 11.7 ± 0.8% over the ensuing 60 min (mean ± SEM, n = 27). Data are reported on a dry-weight (dry wt) basis as previously described (23).

Selection of substrates and their concentrations.

The choice of substrates was based on previous data showing that isolated colonic epithelial cells are capable of activating and oxidizing to CO₂ substrates including acetate, propionate, butyrate, glucose, glutamine and ketone bodies (24–27). In those experiments, the apparent K_m for substrate oxidation was reported to be <1 mmol/L (26–28) and substrate concentrations 5 mmol/L saturated the oxidative pathways with substrate carbon. In these studies, substrate concentrations ranged from 5 to 15 mmol/L. In vivo, butyrate concentrations in cecal fluid and colonic contents of rats, pigs and monkeys were reported to be as low as 3–7 mmol/L when diets containing little or no fermentable dietary fiber were fed, and as high as 40 mmol/L when diets provided ample fermentable fiber (29). In vivo concentrations were reported to vary widely also for propionate (5–45 mmol/L) and acetate (15–380 mmol/L) (29). Glucose and glutamine are most likely to be provided to colonocytes via the arterial blood supply; under fasting conditions, these averaged 4 and 0.5 mmol/L, respectively (30). Ketone bodies, also available to colonocytes via the arterial blood supply during fasting, are largely dependent on the duration of the fast and, in healthy humans, may range from 1 to 8 mmol/L.

Carbon dioxide production.

Aliquots of cell suspension (1 mL) were mixed with Krebs-Henseleit buffer containing ¹⁴C-labeled and unlabeled substrate, incubated and CO₂ collected as previously described (19). Carbon dioxide production from ¹⁴C-butyrate in 5 mmol/L butyrate was measured (19) to ensure normal cellular metabolism, and was 5.60 ± 0.3 µmol CO₂/(g dry wt · min) (mean ± SEM), which is similar to values reported previously.

Inhibition of citrate lyase by (-)-hydroxycitrate.

(-)-Hydroxycitrate is a powerful inhibitor of the enzyme ATP-citrate lyase (31), which catalyzes the cytosolic cleavage of citrate to form oxaloacetate and AcCoA. The acid has also been shown to be an effective inhibitor of fatty acid (21,32–34) and cholesterol synthesis (35–37) in nonintestinal tissues. Citrate lyase was assayed using the method of Srere (38).

Lipid synthesis assay.

Aliquots of cell suspension (1 mL, 4–8 g dry weight/L for ¹⁴C assays, 15–20 g dry weight/L for ³H₂O assays) and incubating media (1 mL) were added in duplicate to 25-mL Erlenmeyer flasks. The incubating media consisted of ¹⁴C-labeled substrate (10–40 kBq/µmol) or ³H₂O (300 mBq/flask) and unlabeled substrates in KH buffer with antibiotics as previously described (19). Flasks were gassed with O₂/CO₂ (19:1, v/v) and sealed with double-seal stoppers. Incubations were performed for 60 min at 37°C in a shaking water bath. After the incubation, reactions were stopped by adding 2 mL methanol.

There were no significant differences in ¹⁴C incorporation into sterols or fatty acids when [U-¹⁴C]acetate, [1-¹⁴C]acetate and [2-¹⁴C]acetate were used as tracers (data not shown), indicating that the isotopes could be used interchangeably. Nonetheless, [1-¹⁴C]acetate was used in all experiments.

Lipid extraction and separation.

Lipids were extracted from the cells with methanol and chloroform according to the method of Bligh and Dyer (39) with modifications by Levin (40). The extracted lipids were applied to TLC plates (20 x 20 cm silica gel GF, Alltech, Deerfield, IL), developed in petroleum ether/ethyl ether/glacial acetic acid (80:20:1), and visualized by spraying with dichlorofluorescein (Sigma, St. Louis, MO). The bands corresponding to cholesterol, lanosterol, phospholipid, free fatty acids (FFA) and triacylglyceride (TAG) standards were scraped from the plate and incubated for 1 h at 40°C in 1 mL of TS-2 tissue solubilizer (Research Products International, Mount Prospect, IL). These bands accounted for >95% of the radioactivity in the lanes for all experiments. At the end of the incubation, 15 mL of 3a70b scintillation cocktail (Research Products International) and 0.105 mL of glacial acetic acid (decrease chemiluminescence) were added to the lipid sample, which was counted using a scintillation counter (Packard, Meridan, CT). When samples were not saponified, incomplete resolution of the cholesterol and phospholipid bands precluded quantitative separation of these two species. For this reason, incorporation values into sterols under nonsaponified conditions were not reported. In all other experiments, radioactivity in total sterols was calculated as the sum of radioactivity in the free cholesterol and lanosterol bands.

In the experiments that measured incorporation into fatty acids [sum of fatty acids present as FFA, in phospholipids (PL) and in TAG], the chloroform extract was saponified in 0.2 mL of 30% alcoholic KOH overnight at room temperature. After saponification, 0.8 mL of water was added and the mixture was acidified with H₂SO₄ (9 mol/L). The lipids were extracted with petroleum ether (3 x 3 mL), blown to dryness under a stream of N₂ and dissolved in chloroform. The lipids were applied to TLC plates and processed as described above.

Recovery of cholesterol and fatty acids was determined in duplicate by adding a known amount of [4-¹⁴C]cholesterol and [1-¹⁴C]palmitate to cell suspension. The incorporation values obtained in the treatment flasks were corrected for any losses in recovery. The values for cholesterol and palmitate recovery were very consistent from flask to flask and day to day.

Calculations.

The incorporation of ³H₂O into lipids was converted from Bq/min to µmol by dividing by the specific activity (Bq/µmol) of the substrate using ^{Eq. (1)}.

(1)

Tritiated water incorporation data are reported as µmol ³H₂O/(g dry wt · h).

The incorporation of ¹⁴C-substrates into lipids was converted from Bq to nmol by dividing by the specific activity (Bq/µmol) of the substrate using ^{Eq. (2)}.

(2)

¹⁴C-Substrate incorporation data are reported as nmol substrate carbon/(g dry wt · h). The recovery of cholesterol and palmitate was 85.3 ± 0.8 and 82.5 ± 1.3% (means ± SEM, n = 26), respectively. To calculate the results as nmol substrate carbon, it was necessary to convert the result from ^{Eq. (2)} into nmol AcCoA carbon incorporated followed by conversion to nmol substrate carbon.

To calculate substrate incorporation into fatty acids, values determined in ^{Eq. (2)} were multiplied by the number of carbons in the substrate that could be incorporated into fatty acids. Because none of the carbons of acetate, butyrate and 3-hydroxybutyrate are lost in their incorporation into fatty acids, the value determined in ^{Eq. (2)} was multiplied by 2, 4 and 4, respectively. The incorporation of propionate, glucose and glutamine into fatty acids results in the loss of some carbons as CO₂; thus the value determined in ^{Eq. (2)} was multiplied by 2, 4 and 2, respectively.

To calculate nmol carbon incorporated into sterols from nmol substrate, it was necessary to first convert to nmol AcCoA incorporated to correct for the unequal losses of the number one and two carbons of AcCoA in the cholesterol synthetic pathway. When the radioactive label was on the number one carbon, ^{Eq. (3)} was used.

(3)

The factor 15/12 was used to calculate incorporation of the number two carbon of AcCoA. This was done by multiplying the measured incorporation of the number one carbon by 15/12, because 12 of the 27 carbons of cholesterol are from the number one carbon of AcCoA and 15 are from the number two carbon.

To calculate nmol substrate carbon incorporated into sterols from nmol AcCoA, a second correction factor that indicates the nmol substrate carbon/nmol AcCoA carbon was introduced into these calculations. Nanomoles of substrate can be converted directly to nmol of substrate carbon by multiplying the incorporation rates by the number of carbon atoms in each substrate that will ultimately form AcCoA. For acetate, butyrate, propionate, 3-hydroxybutyrate and citrate, the values calculated from the above equations were multiplied by 2/2, 4/2, 2/2, 4/2 and 2/2 nmol substrate carbon/nmol AcCoA carbon, respectively. CO₂ production was calculated as previously described (19).

Statistical analysis.

Data are presented as means ± SEM. Differences were determined using paired t tests and ANOVA (two-way and repeated measures). When present, differences were identified using Tukey’s Studentized range test. Differences were considered significant at P < 0.05. Computer programs were used to perform computations (41,42).

	RESULTS

TOP ABSTRACT MATERIALS AND METHODS RESULTS DISCUSSION LITERATURE CITED

 
De novo synthesis of fatty acids.

There was net synthesis of fatty acids by rat colonocytes as indicated by incorporation of ³H₂O into all three classes of fatty acid–containing lipids (Fig. 1). Rat colonocytes incorporated 9.15 ± 0.11 µmol ³H₂O/(g dry wt · h) into fatty acids. Significantly more label (61%) was incorporated into phospholipid than into TAG or FFA. Similarly, significantly more ¹⁴C-acetate was incorporated into phospholipid than into FFA or TAG when cells were incubated with ¹⁴C-acetate.

View larger version (17K): [in this window] [in a new window]   FIGURE 1 Incorporation of 3H2O and acetate carbon into free fatty acids (FFA), phospholipids (PL) and triacylglycerides (TAG) by rat colonic epithelial cells. Cells were incubated in 15 mmol/L acetate, propionate, butyrate, glucose, and glutamine with 3H2O (300 MBq) or with [1-14C]acetate (18.5 Bq/µmol). In the 14C experiments, 10 mmol/L 3-hydroxybutyrate was included also in the incubation media. Values are means ± SEM, n = 6. Data were analyzed by one-way ANOVA (P values were < 0.05 for data in both panels); differences were evaluated by Tukey’s Studentized range test. Bars with different letters differ.

Into both sterols and fatty acids, significantly more carbon was incorporated from acetate than from the other substrates that were evaluated in an equimolar mix of five substrates (Table 1). Incorporation of butyrate carbon was intermediate. Very small and significantly lower levels of carbon incorporation into sterols and fatty acids were observed from propionate, glucose and glutamine.

(-)-Hydroxycitrate concentrations from 0 to 7 mmol/L had no effect on CO₂ production from [1-¹⁴C]butyrate in 5 mmol/L butyrate or on LDH leakage in isolated colonocytes (data not shown). Because there was no evidence of toxicity, (-)-hydroxycitrate was used at 7 mmol/L in all subsequent experiments to allow for maximal inhibition of citrate lyase. Under baseline conditions, the citrate lyase activity of cell preparations were 0.73 ± 0.01 µmol/(g dry wt · min). (-)-Hydroxycitrate at 7 mmol/L reduced the activity of colonocyte citrate lyase by 86.6 ± 0.96% (Fig. 2). Evidence that hydroxycitrate inhibited citrate lyase activity in intact colonocytes was provided by measuring oxidation and incorporation into lipids of exogenous citrate. Hydroxycitrate at 7 mmol/L had no influence on CO₂ production from citrate but significantly reduced incorporation of citrate carbon into both cholesterol and fatty acids (Table 2).

View larger version (21K): [in this window] [in a new window]   FIGURE 2 Influence of hydroxycitrate on citrate lyase activity in homogenates, and on incorporation of acetate or butyrate into sterols and fatty acids by rat colonic epithelial cells. Citrate lyase activity was determined on aliquots of cell supernatants. Incorporation of substrate into lipids was assessed using intact cells incubated with [1-14C]acetate in 10 mmol/L acetate and glucose, or with [1-14C]butyrate in 10 mmol/L butyrate and glucose. Values are means ± SEM, n = 3. Data were analyzed by paired t tests. *Indicates that values with and without (-)-hydroxycitrate (0 vs. 7 mmol/L) differ, P < 0.001.

Role of ketone bodies in the lipogenesis pathways of colonocytes.

To determine whether ketone bodies, acetate and butyrate form a common biosynthetic precursor pool for lipid synthesis, we evaluated the effects of ketone bodies on SCFA incorporation into lipids. Both 3-hydroxybutyrate and acetoacetate significantly suppressed incorporation of butyrate carbon into sterols and fatty acids and suppression was greater for acetoacetate than for 3-hydroxybutyrate (Fig. 3). 3-Hydroxybutyrate suppressed incorporation into sterol and fatty acids by 35 and 27%, respectively. Acetoacetate suppressed incorporation into sterols and fatty acids by 59 and 48%, respectively. By contrast, acetoacetate did not significantly suppress incorporation of acetate carbon into either sterols or fatty acids.

View larger version (22K): [in this window] [in a new window]   FIGURE 3 Influence of ketone bodies on incorporation of butyrate and acetate carbon into sterols and fatty acids by rat colonic epithelial cells. Upper panel: Butyrate and tracer ([1-14C]butyrate, 15 mBq/µmol), glucose and, where indicated, 3-hydroxybutyrate or acetoacetate were present at 10 mmol/L. Lower panel: acetate and tracer ([1-14C]acetate, 10 Bq/µmol), glucose and, where indicated, acetoacetate were present at 10 mmol/L. Bars represent means ± SEM, n = 3. After log transformation, data were analyzed using one-way ANOVA. P-values for the upper panel were 0.002 for sterols and 0.005 for fatty acids. Within each data set, treatments with different letters differ, P < 0.05.

	DISCUSSION

TOP ABSTRACT MATERIALS AND METHODS RESULTS DISCUSSION LITERATURE CITED

TCA cycle–generated AcCoA does not appear to play a major role in providing precursors for lipid biosynthesis in colonic epithelial cells. As evidence, negligible incorporation into lipids was observed for glucose, glutamine and propionate, whereas incorporation was intermediate for butyrate and highest for acetate when cells were presented with an equimolar mixture of these substrates. By contrast, we observed previously that carbon dioxide production was greatest from butyrate, intermediate from glucose and glutamine, and least from acetate when assessed in colonocytes prepared using conditions identical to those of the present experiment (24). If TCA cycle entry followed by citrate efflux is the major route for AcCoA transport of precursor from the mitochondria to the cytosol for the purpose of lipid synthesis, one would expect that relative conversion of substrates to CO₂ should be similar to relative incorporation into lipids. This was clearly not the case.

Evidence that the TCA cycle plays a minor role in providing precursors for lipid synthesis is provided also by experiments using hydroxycitrate, an inhibitor of cytosolic ATP-citrate lyase. Although we showed that hydroxycitrate effectively suppressed colonocyte citrate lyase and inhibited incorporation of citrate into sterols and fatty acids, hydroxycitrate did not suppress incorporation of either acetate or butyrate into sterols or fatty acids. If acetate- or butyrate-derived acetyl units were transported from the mitochondria to the cytosol predominantly via citrate, hydroxycitrate would be expected to suppress incorporation of acetate and/or butyrate carbon into lipids. This was not observed. Thus, the TCA cycle does not play a major role in providing synthetic precursors for lipogenesis in colonocytes. The lack of an effect by hydroxycitrate on acetate incorporation into lipids agrees well with observations in rat liver (33) and brain (32), where acetate may be activated in the cytosol. Although this might provide an explanation regarding the pathway by which acetate is incorporated into lipids in colonocytes, butyrate is not known to be activated in any compartment other than the mitochondria. In hepatocytes, hydroxycitrate prevents incorporation of butyrate carbon into fatty acids (34). Our data suggest that the transport of butyrate carbon via citrate occurs to a far lesser extent in colonocytes than in hepatocytes. A possible alternative to acetyl unit transport by citrate would be transfer via the ketone bodies.

Butyrate and ketone bodies appear to constitute a common precursor pool for lipid synthesis.

Carbon from 3-hydroxybutyrate was incorporated into sterols and fatty acids and incorporation of butyrate carbon into lipids was significantly decreased by both 3-hydroxybutyrate and acetoacetate. By contrast, acetoacetate did not suppress acetate incorporation into lipids. These results suggest that butyrate and ketone bodies contribute to a common synthetic precursor pool. The presence of mitochondrial HMG-CoA synthase in rat colonic mucosa was reported by others (43) and may be required for metabolism of SCFA to ketone bodies.

Cytosolic activation of acetate.

Acetate appears to be the preferred carbon source for lipogenesis in isolated colonocytes, because when present at equimolar concentrations together with several key substrates, acetate contributed more carbon to the synthesis of lipid than any other substrate tested. Butyrate carbon incorporation into lipid was suppressed by >50% when other substrates were available (Table 3), whereas acetate carbon incorporation into lipid was similar in the presence (Table 1) and absence (Fig. 3) of alternative substrates. In contrast, the metabolism of butyrate to CO₂ was not influenced by the presence of alternative substrates, whereas the metabolism of acetate to CO₂ was (24). This suggests important differences in the pathways and pools involved in metabolism of acetate and butyrate to CO₂ vs. sterols and fatty acids.

Several observations support the suggestion that acetate and butyrate do not form a single, common, precursor pool for the synthesis of lipids. In previous studies, using protocols and mixtures of substrates comparable to those used in the present study, the ratio of acetate/butyrate for incorporation into CO₂ was 1:8 (24). Also in colonocytes, incorporation into ketone bodies was found to be considerably lower from acetate than from butyrate [1:10 (44)]. By contrast, the ratio of acetate/butyrate incorporation into lipids was 2:1 in this study. Because the acetate and butyrate molecules must be activated in the mitochondria to be metabolized to CO₂ and ketone bodies, and because both CO₂ and ketone bodies are produced from acetate to a far lesser extent than from butyrate when available simultaneously, acetate may be activated in the mitochondria to a considerably lower extent than butyrate. Our observation that acetate makes a larger contribution than butyrate to lipid synthesis can be explained only by invoking a second precursor pool to which acetate makes a major contribution. It is possible that incorporation of acetate into newly synthesized lipids relies on activation in both the cytosol and mitochondria of colonic epithelial cells as occurs in the liver (33) and brain (32).

Fatty acid classes.

The incorporation of ³H and ¹⁴C was greater into PL than into TAG and FFA. To a similar extent in colonic epithelium, much more lipid is present in PL than in either TAG or FFA (45). Unlike the enterocytes of the small intestine, colonocytes do not secrete lipoproteins, which may explain why the esterification of FFA to TAG was relatively small compared with the incorporation of FFA into PL. Due to the smaller ³H and ¹⁴C incorporation into TAG relative to PL, the main role of fatty acid synthesis in the colonocyte may be the provision of PL for membrane assembly.

In summary, these data suggest that colonocytes synthesize lipids in a manner that is distinct from that of other tissues such as liver. Unlike the liver, citrate transport does not appear to be very important in colonocytes. Butyrate and ketone bodies appear to form a common lipid synthetic precursor pool in colonocytes, suggesting that ketone bodies are a possible alternative for transport of acetyl units to the cytosol. Under the conditions evaluated, acetate and butyrate were the major carbon contributors to lipids in colonocytes and, although not directly tested, acetate may be activated to a great extent in the cytosol followed by direct incorporation into lipids. The high proportion of synthesized fatty acids found in phospholipid suggest that the major function of lipid synthesis in the epithelial cells of the colon is for membrane assembly.

	FOOTNOTES

 
¹ Presented in part at Experimental Biology 97, April 1997, New Orleans, LA [Zambell, K. L. & Fleming, S. E. (1997) Comparison of the relative incorporation rates of ¹⁴C-labeled substrates into lipids in rat colonocytes. FASEB J. 11: A382 (abs.)].

² Supported by the Agriculture Experiment Station and the National Institutes of Health (AG-10765).

⁴ Abbreviations used: AcCoA, acetyl-CoA; FFA, free fatty acids; HMG-CoA, hydroxymethyl glutaryl CoA; LDH, lactate dehydrogenase; PL, phospholipid; TAG, triacylglyceride; TCA, tricarboxylic acid.

Manuscript received 13 May 2003. Initial review completed 7 July 2003. Revision accepted 20 August 2003.

	LITERATURE CITED

TOP ABSTRACT MATERIALS AND METHODS RESULTS DISCUSSION LITERATURE CITED

 

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