Intralipid

ABSTRACT. Background: Supplementation of total parenteral nutrition (TPN) with a mixture of short-chain fatty acids (SCFA) enhances intestinal adaptation in the adult rodent model. However, the ability and timing of SCFA to augment adaptation in the neonatal intestine is unknown. Furthermore, the specific SCFA inducing the intestinotrophic effects and underlying regulatory mechanism(s) are unclear. Therefore, we examined the effect of SCFA supplemented TPN on structural aspects of intestinal adaptation and hypothesized that butyrate is the SCFA responsible for these effects. Methods: Piglets (n = 120) were randomized to (1) control TPN or TPN supplemented with (2) 60 mmol/L SCFA (36 mmol/L acetate, 15 mmol/L propionate and 9 mmol/L butyrate), (3) 9 mmol/L butyrate, or (4) 60 mmol/L butyrate. Within each group, piglets were further randomized to examine acute (4, 12, or 24 hours) and chronic (3 or 7 days) adaptations. Indices of intestinal adaptation, including crypt-villus architecture, proliferation and apoptosis, and concentration of the intestinotrophic peptide, glucagon-like pepide-2 (GLP-2), were measured. Results: Villus height was increased (p

More than 10,000 patients in the United States have short-bowel syndrome (SBS),1 which is defined as intestinal failure resulting from massive small bowel resection.2 Conditions that need surgical intervention leading to SBS in the pediatrie population include necrotizing enterocolitis, intestinal atresias, extensive intestinal aganglionosis, and abdominal wall defects such as gastroschisis and malrotation with volvulus.3-8 Immediately after intestinal resection, the process of intestinal adaptation begins in all layers of the intestinal wall.9-11 The most notable response occurs in the residual ileum,9-11 with increases in enterocyte proliferation as soon as 8 hours,12 reinforcing the rapidity in which the remnant segment responds to the intestinal resection. However, before adaptation is complete, many patients with SBS have intractable diarrhea, steatorrhea, weight loss, electrolyte abnormalities, metabolic imbalances, and multiple nutritional deficiencies that lead to malnutrition.13 Thus, the provision of total parenteral nutrition (TPN) is necessary, as it is instrumental in meeting the nutritional needs and maintaining normal growth and development for children with SBS.8 However, infants may be at increased risk for intestinal insufficiency because the marked structural and functional maturation that occurs in the gastrointestinal tract during the perinatal period is inhibited with TPN,14'15 presumably because of the absence of enterai stimulation.16 Indeed, approximately 30% of children with SBS are dependent on receiving TPN,17 and many others experience delayed physical development and possible cognitive deficits related to nutritional deficiencies.18 Clearly, therapies aimed at stimulating structural and functional adaptations in the rapidly developing intestine are vital for infants with SBS.

Supplementation of the short-chain fatty acids (SCFA) acetate, propionate, and butyrate to TPN reduces parenteral nutrition-induced atrophy19 and stimulates both structural and functional aspects of intestinal adaptation after surgical resection.20-23 However, this research was performed in the adult rat; hence, the effects of SCFA in the neonatal intestine are relatively unknown. Additionally, it is not known if all 3 SCFA are required for the intestinal effects reported previously, as provision of butyrate alone has been shown to increase mucosal growth and epithelial proliferation in both the small and large intestine.24,25 Moreover, the time course of SCFA effects in enhancing intestinal adaptation in the neonatal intestine has not been explored; thus, delineation of acute and chronic effects is needed. Finally, there is evidence that systemic SCFA and dietary fiber stimulate the secretion of the intestinotropic peptide glucagon-like peptide-2 (GLP-2).21,23,26-28 Therefore, additional research is necessary to examine potential trophic stimuli(us), temporal modifications and regulatory mechanism(s) involved with structural indices of intestinal adaptation.

Our study was designed to determine the efficacy of TPN supplemented with SCFA in a clinically relevant, neonatal model that focused on the following 4 pertinent questions:

1. In the neonatal intestine, do SCFA enhance structural aspects of intestinal adaptation by increasing proliferation and decreasing apoptosis?

2. Are the intestinotrophic effects caused by the presence of all 3 SCFA (acetate, propionate, and butyrate), just the butyrate portion of the SCFA mixture (9 mmol/L), or the total dose of SCFA (60 mmol/L)?

3. How rapidly do SCFA augment structural aspects of intestinal adaptation, and are the effects sustained through chronic time points?

4. Is GLP-2 a potential mechanism mediating the trophic effects of SCFA?

We hypothesized that supplementation of TPN with butyrate alone would rapidly increase structural indices of intestinal adaptation in the neonate and that these adaptive responses would be associated with increased plasma GLP-2 concentration. In order to examine these hypotheses, we used the neonatal piglet to simulate the TPN-supported human infant with SBS due to the similarities in nutritional requirements, gastrointestinal physiology, and metabolism.29,30

MATERIALS AND METHODS

Experimental Design

Neonatal piglets (n = 120) were obtained from the Imported Swine Research Laboratory at the University of Illinois at Urbana-Champaign within 48 hours of birth and underwent a superior vena cava cannulation, swivel placement and 80% proximal jejunoileal resection. Piglets (1.77 ± 0.16 kg, n = 120) were randomized according to initial body weight to (1) TPN (control) or TPN supplemented with (2) 60 mmol/L SCFA (36 mmol/L acetate, 15 mmol/L propionate and 9 mmol/L butyrate; SCFA), (3) 9 mmol/L butyrate (9Bu), or (4) 60 mmol/L butyrate (60Bu). Within each group, animals were further randomized to examine acute (4, 12, or 24 hours) and chronic (3 or 7 days) adaptations.

Surgical Procedures: Central Line Placement and Massive Small Bowel Resection

The protocol was approved by the Laboratory Animal Care Advisory Committee at the University of Illinois at Urbana-Champaign. Sterile instruments and aseptic technique were used at all times. Piglets were placed under general anesthesia (98% oxygen/2% isoflurane; Baxter Pharmaceutical Products, Inc, Deerfield, IL). The surgical areas around the external jugular and abdomen were aseptically prepared with betadine (10% povidone-iodine; Purdue Frederick Co, Norwalk, CT), and locally anesthetized using lidocaine (2% lidocaine HCl, 20 mg/mL; Abbott Laboratories, North Chicago, IL). A 3 cm incision was made in the right clavicle region to isolate the external jugular for insertion of a catheter (3.5 French polyvinyl chloride catheter; Bergen-Brunswig, Lake Zurick, IL). The catheter was inserted 6 cm through the external jugular into the superior vena cava for infusion of TPN. The catheter was flushed with approximately 5 mL of heparinized saline (20 LVmL; Sigma Chemical Co, St. Louis, MO) and tunneled subcutaneously for externalization within the subscapular region.

All animals were also subjected to an 80% proximal jejunoileal resection, as previously described by Tappenden and colleagues.2-23,26 A laparotomy was performed and 15 cm of jejunum distal to the ligament of Treitz and 75 cm of ileum proximal to the ileocecal junction were measured using sterile silk ribbon placed along the antimesentric border of the gently stretched small intestine. Intestine not included in the measurement was excised after vessel cauterization. Bowel continuity was restored by an end-to-end jejunoileal anastomosis with interrupted 5-0 silk sutures (Ethicon, Inc, Somerville, NJ). Thus, all animals were left with an equivalent length of proximal jejunum and distal ileum. The abdomen was closed with interrupted 3-0 silk sutures (Ethicon, Inc).

Piglets were monitored for activity level throughout the recovery period to ensure proper recovery from surgery. Piglets received Naxcel antibiotic (3 mg/kg body weight; SmithKline Beecham Corp, Philadelphia, PA) before surgery and during the entire study period. Buprenorphine analgesic (0.01 mg/kg body weight; Henry Schein Inc, Melville, NY) was administered intramuscularly every 12 hours for the first 24 hours postsurgery to minimize pain and discomfort inherent to any surgical procedure.

Animal Care and Housing

After surgery, piglets were fitted with jackets to which a swivel tether (Alice King Chatham Medical Arts, Hawthorne, CA) was attached to protect the catheter and infusion lines and allow for free mobility of the piglets. Piglets were housed individually in clean metabolic cages (approximately L 76 cm × W 23 cm × H 46 cm) located at the Edward R. Madigan Laboratory animal care facility at the University of Illinois at Urbana-Champaign. HEPA-filtered airflow to each suite was individually controlled and the room temperature was maintained at 30°C with a 12-hour light/dark cycle. Additional heat was provided to the piglets by radiant heaters located on top of the cages to maintain a local temperature of ~34°C. A full clinical assessment was performed every morning according to the following criteria: weight gain, body temperature, respiration rate, activity level, healing of surgical site, and absence of edema and guarded posture. A partial clinical assessment was performed each evening to reevaluate the clinical outcomes listed except body weight.

Nutrient Solutions

The TPN solutions were prepared daily under a laminar flow hood to maintain sterility and were filter sterilized before infusion (0.22 µm milliporefilter; Millipore Corporation, Bedford, MA). The solutions were provided as a 3-in-1: dextrose (50% glucose monohydrate; Baxter Healthcare Corporation), amino acids (8.5% Travasol with electrolytes; Baxter Healthcare Corporation) and lipids (30% Intralipid; Baxter Healthcare Corporation). These were contained in the same infusion solution in addition to vitamins, minerals and SCFA. Compositions of the TPN solutions are listed in Table I. TPN was continuously infused via IMED 980 volumetric infusion pumps (St. Louis, MO). Because of surgical stress related to the intestinal resection, energy and protein needs were estimated to be increased by 20%; thus, for the first 3 postoperative days parenteral nutrition solutions provided 307 kcal/kg per day and 15.5 g amino acids/kg per day, and 253 kcal/kg per day and 12.8 g/kg per day for postoperative days 4 to 7, with a nonprotein kilocalorie-tonitrogen ratio of 100:1. The SCFA-acetate, propionate, and butyrate-were added as free acids (Sigma Chemical Co) in the molar proportions found physiologically in the colon.31 The SCFA supplements provided approximately 2 (SCFA), 0.6 (9Bu) and 3.3% (60Bu) of kcal contained within 1 L of control TPN solution and replaced an equivalent amount of dextrose to ensure isoenergic formulations.

Sample Collection

After the study period, 2.5 g sodium pentobarbital (Fatal Plus; Veterinary Laboratories, Inc, Lenexa, KS) was administered IV to each piglet. Blood samples drawn by cardiac puncture were collected into prechilled vacutainers containing EDTA (0.3 mg; Fisher Scientific, Itasca, IL) and 2 U TIU of aprotinin (trypsin inhibitor units; Sigma Chemical Co). Plasma was separated by centrifugation at 3500 × g for 15 minutes at 4°C. Plasma was then snap-frozen in liquid nitrogen, and stored at -70°C until further use. The gastrointestinal tract was rapidly removed. The duodenum was excised proximal to the ligament of Treitz and distal of the stomach. The intestine distal to the ligament of Treitz and proximal to the anastomosis was designated jejunum, whereas ileum included the remaining intestine proximal to the ileocecal valve. The entire colon was removed distal to the ileocecal valve. Segments were flushed with ice-cold saline, weighed and length was measured by suspending the intestine longitudinally with a 10 g weight attached to the end. Samples from each segment were fixed in 10% buffered formalin for histomorphological assessment, scraped for quantification of mucosal and submucosal dry weight, and snap-frozen in liquid nitrogen and stored at -70°C for analysis of DNA, RNA, protein and Western immunoblots.

DNA, RNA and Protein Content

Intestinal samples were homogenized (model PCU11; Brinkman Instruments, Westbury, NY) and sonicated for 60 seconds at setting 3 (model 450; Branson Sonifier, Danbury, CT) in autoclaved diethyl pyrocarbonate (DEPC; 1 mL/L; Sigma Chemical Co) -treated distilled water. The intestinal homogenates were used to determine the intestinal DNA and RNA concentrations using a modified Prasad method.32 Briefly, black 96-well plates were prepared by adding 7.5 µL of diluted (1:20 with DEPC water) intestinal homogenate, 142.5 µL of buffer (0.1 M Tris, 0.1 M NaCl), and 150 µL ethidium bromide (10 mg/mL; Sigma Chemical Co) to each well. Intestinal nucleotide concentration was determined fluorometrically at an excitation of 522 nm and an emission of 610 nm using a SPECTRAmax Gemini XS (Molecular Devices, Sunnyvale, CA). An initial fluorescence reading was taken to determine the total nucleotide concentration in the well. Three µl of RNAase Type III-A (20 mg/mL; Sigma Chemical Co) was then added to each well, followed by incubation in a hybridization oven at 50°C for 1 hour. A final fluorescence reading was obtained at room temperature. The final RNA concentration was determined using standard-curve methodology with both herring sperm DNA (Promega Corporation, Madison, WI) and RNA Type III from bakers' yeast (Sigma Chemical Co). The curve concentration ranged from 0 to 35 µg/mL, and an equal amount of each standard was added to the well to obtain the final desired concentration. The initial fluorescence reading taken was the total nucleotide concentration curve and the final fluorescence reading was the DNA concentration curve. Therefore, the RNA concentration curve was calculated by subtracting the total nucleotide curve from the DNA curve. Total protein concentrations were determined using the Bio-Rad Protein Assay (Bio-Rad, Hercules, CA) with an IgG standard33 measured at 595 nm using an El^sub x^800 plate reader (Bio-Tek, Winooski, VT).

Histomorphology

Formalin-fixed intestinal samples were embedded in paraffin, sliced to approximately 5-µm thickness with a microtome and stained with hematoxylin and eosin. Villus height, midvillus width, and crypt depth were measured by using a Nikon Optiphot-2 microscope (Nikon, Melville, NY) and Image-Pro Express software (Version 4.5; Media Cybernetics, Inc, Silver Spring, MD) in 8 to 10 well-oriented villi and crypts. Villus surface area (villus height × midvillus width) was also calculated. In addition, intestinal segment circumference was measured to estimate intestinal surface area.

Intestinal Cell Proliferation

Proliferating cell nuclear antigen (PCNA) protein abundance was determined in tissue homogenate. Total protein concentrations were determined using the Bio-Rad Protein Assay with an IgG standard. Protein aliquots were denatured by boiling for 4 minutes, separated by size using 15% SDS-PAGE and were transferred to polyvinylidene difluoride (PVDF) membranes (Bio-Rad) using a Trans Blot SD Semi-Dry Electrophoretic Transfer Cell (Bio-Rad). Before analyzing experimental samples, a linear range from O to 30 µg was established as the appropriate amount of protein per well to ensure that bands produced fell within the linear range of the colorimetric detection system. Western blot analysis for PCNA (8 µg duodenum and colon; 12.5 µg jejunum and ileum) protein was performed using a monoclonal antibody (Chemicon International, Temecula, CA). PVDF membranes were developed using the Opti-4CN kit (Bio-Rad). Briefly, PVDF membranes were blocked using 5% nonfat dry milk in phosphate-buffered saline with Tween 20 (PBST; 4.3 mmol/L Na^sub 2^PO^sub 4^, 1.4 mmol/L KH^sub 2^PO^sub 4^, 2.7 mmol/L KCl, 137 mmol/L NaCl, 0.1% Tween 20, pH 7.3) for 3 hours at room temperature on a metabolic shaker. The membranes were quickly rinsed with PBST to remove excess blocking buffer. The PCNA (36 kDa) primary antibody was diluted 1:2500 in PBST containing 0.01% bovine serum albumin (BSA; Sigma Chemical Co) and incubated for 2 hours after washes with PBST at room temperature (6 × 5 minutes). Secondary antibody goat antimouse IgG conjugated to horseradish peroxidase was incubated at room temperature for 1 hour (1:10,000 dilution in PBST containing 0.01% BSA; BioRad) and washed with PBST at room temperature (3 × 10 minutes). Colorimetric detection followed addition of Opti-4CN diluent solution. Membranes were photographed using the Kodak ID Image Station (Eastman Kodak Company, Rochester, NY). Densitometry of PCNA protein abundance was performed using Kodak ID Image Analysis Software (Version 3.5.4; Eastman Kodak Company).

Epithelial Cell Proliferation

The immunohistochemical detection of PCNA was used as an index of the rate of crypt cell proliferation. Briefly, formalin-fixed, paraffin-embedded sections sliced to approximately 5-µm thickness were cleared of paraffin with xylene and then rehydrated with a series of ethanol washes (100%, 95%, 80% and 70%). Fixed proteins were digested with 0.1% trypsin (Sigma Chemical Co) at 37°C for 15 minutes, and endogenous peroxidase activity was quenched with 5% hydrogen peroxide (Fisher Scientific) for 15 minutes. Slides were washed with PBS at room temperature (3×5 minutes) after trypsin and hydrogen peroxide treatment. The slides were then blocked with 1.5% normal horse serum (NHS; Vector Laboratories, Burlingame, CA) for 30 minutes. The PCNA primary antibody (Chemicon International) was diluted 1:100 in phosphate-buffered saline (PBS; 2 mmol/L NaH^sub 2^PO^sub 4^.H2O, 8.5 mmol/L Na^sub 2^HPO^sub 4^, 150 mmol/L NaCl, pH 7.5) with 0.01% BSA (Sigma Chemical Co). The primary antibody was incubated for 1 hour at room temperature and washed with PBS at room temperature (4×5 minutes). A biotinlyated universal secondary antibody was diluted 1:200 in PBS with 1.5% NHS and incubated for 30 minutes at room temperature followed by a 30-minute incubation with the ABC complex after manufacturer instructions (Elite Vectastain ABC kit; Vector Laboratories). PCNA-labeled cells were visualized using 3,3'-diaminobenzidine (Vector Laboratories). The slides were then washed in PBS, dehydrated in ethanol (70%, 80%, 95% and 100%) and xylene, and then coverslipped. The number of PCNA-labeled nuclei in 8 to 10 well-oriented crypts was counted using a Nikon Optiphot-2 microscope (Nikon) and Image-Pro Express software (Version 4.5; Media Cybernetics, Inc), this number was used to quantify the number of proliferating crypt cells.

Apoptotic Signaling Proteins

Apoptotic protein abundance was quantified using Western blot analysis in tissue homogenate. Bax, a pro-apoptotic protein, was measured in the entire length of the intestine; however, because of regionspecific differences in the regulation of apoptosis in the intestine, the antiapoptotic proteins Bcl-w and Bcl-2 were measured in the small intestine and colon, respectively. Protein abundance was quantified according to the previous SDS-PAGE protocol, with the following exceptions: protein aliquots for Bax (10 µg duodenum; 15 µg jejunum, ileum and colon), Bcl-w (10 µg jejunum; 8 µg duodenum and ileum), and Bcl-2 (15 µg colon) were separated by size using 18% SDS-PAGE, and the Bax (21 kDa), Bcl-w (21 kDa), and Bcl-2 (25 kDa) primary antibodies (Chemicon International) were diluted 1:2500 and expressed as a ratio of proapoptotic to antiapoptotic protein.

Plasma GLP-2 Concentrations

Plasma GLP-2 concentration was quantified in collaboration with Jens J. Hoist at the University of Copenhagen, Panum Institute, Copenhagen, Denmark.34 Briefly, plasma samples were extracted with 75% ethanol and centrifuged at 3000 × g for 30 minutes at 4°C. The supernatant was decanted, lyophilized, and reconstituted to the original plasma volume in assay buffer (80 mmol/L Na^sub 3^PO^sub 4^^sup -^, 0.01 mmol/L valine-pyrrolidide, 0.1% wt/vol human serum albumin, 10 mmol/L EDTA, 0.6 mmol/L thimerosal, pH 7.5). Approximately 300 µL of extracted samples and human GLP-2(1-33) standards were incubated with 100 µL of rabbit GLP-2 antiserum (final dilution 1:25,000) for 24 hours at 4°C, after which free and bound peptides were separated by absorption to plasma-coated charcoal. This antiserum is raised against the NH^sub 2^ terminal fragment of human GLP-2 and specifically recognizes the NH^sub 2^ terminal region of both human and porcine GLP-2.

Statistical Analysis

Statistical analyses were carried out by analysis of variance (ANOVA) for a split-plot design.35 Sources of variation were block (litter), time (main plot), treatment (subplot), and treatment × time. Treatment and time comparisons were evaluated using orthogonal single df comparisons. Treatment comparisons (3 df) were control vs supplemented TPN treatments, SCFA vs butyrate, and 9Bu vs 60Bu; time comparisons (4 df) were acute (4, 12 and 24 hours) vs chronic (3 and 7 days), 4 hours vs 12 hours and 24 hours, 12 hours vs 24 hours and 3 days vs 7 days. Also, certain nonorthogonal single df comparisons of interest were evaluated. If a significant treatment × time interaction (12 df) existed, the interaction was broken down into its component parts. Because of the variation in development intensity of immunoblots, Westerns blots were analyzed statistically as differences from littermate control values. Computations were conducted using the mixed model analysis in SAS (Version 8.2; SAS Institute, Gary, NC). Statistical significance was defined as p

RESULTS

Nutritional Support

All piglets received continuous infusion of nutrients throughout the study period. Nutrient solutions were infused at a rate to provide adequate growth and meet nutritional needs. Initial body weight (1.77 ± 0.16 kg) did not differ among groups. Daily weight gain (0.15 ± 0.01 g/day) did not differ significantly among groups (data not shown). In addition, organ weights did not differ at time of euthanization (data not shown).

Gross Structural Adaptations

Gross intestinal structure was evaluated through measurements of intestinal weight, length and mucosal dry weight. Duodenal weight (mg/cm^sup 2^/kg BW) did not differ among treatments, but mucosal weight was increased (p = .050) by the 60Bu treatment compared with the other treatments, irrespective of time (control = 72 ± 2.3; SCFA = 74 ± 2.3; 9Bu = 73 ± 2.3; 60Bu = 82 ± 2.3 mucosa/total weight). Jejunal weight (mg/cm^sup 2^/kg BW) was increased (p = .003) in the 9 mmol/L butyrate treatments (SCFA and 9Bu) compared with the 60Bu group, regardless of time (SCFA = 43 ± 1.8; 9Bu = 46 ± 1.8; 60Bu = 39 ± 1.8 mg/cm^sup 2^/kg BW). All 3 supplemented TPN treatments (SCFA, 9Bu, and 60Bu) increased (p = .004) ileal weight compared with control (control = 40 ± 1.8, SCFA = 44 ± 1.8, 9Bu = 47 ± 1.8, 60Bu = 42 ± 1.8 mg/cm^sup 2^/kg BW), regardless of time. In addition, mucosal weight was increased in the ileal 9Bu group (p = .019) by 6% compared with the control and was greater (p = .008) than the 60Bu group, regardless of time (control = 72 ± 1.9, SCFA = 72 ± 1.9, 9Bu = 77 ± 1.9, 60Bu = 72 ± 1.9 mucosa/total weight). No effect of treatment on intestinal weight or length was noted in the colon. Thus, regardless of time, butyrate supplemented TPN increased gross indices of intestinal structure most notably in the residual ileum.

Cellular Composition Adaptations

The effect of intestinal adaptation at the cellular level was assessed through the analysis of intestinal DNA, RNA and protein concentration. No effect of treatment on DNA concentration was noted in the duodenum, jejunum, or colon (Table II). However, DNA concentration (µg/cm^sup 2^ tissue) was increased (p = .004) equally in the ileum by the supplemented TPN treatments (SCFA, 9Bu, and 60Bu), irrespective of time (Table II). Regardless of treatment, duodenal cellular RNA concentration was increased (p = .001) at chronic (3 and 7 days) time points compared with acute time points (4, 12, and 24 hours; 4 hours = 0.67 ± 0.10, 12 hours = 0.77 ± 0.10, 24 hours = 0.99 ± 0.10, 3 days = 1.01 ± 0.10, 7 days = 1.29 ± 0.10 µg RNA/µg DNA). Cellular RNA concentration (µg RNA/µ DNA) in the jejunum or ileum did not differ with treatment or time, whereas colonie cellular RNA was consistently increased (p = .003; Table II) by the supplemented TPN treatments (SCFA, 9Bu and 60Bu) compared with the control at all time points. No treatment effect was noted in any intestinal segment for cellular protein concentration. Therefore, it can be concluded that butyrate treatments increased the number of cells in the residual ileum, and also increased the amount of RNA per cell in the colon at all time points examined.

Crypt-Villus Architecture Adaptations

Assessment of epithelial adaptation was completed through histologie examination of intestinal crypt-villus architecture. Supplemented TPN treatments significantly increased crypt-villus architecture. Duodenal villus height was increased (p

In general, the supplemented TPN treatments (SCFA, 9Bu, and 60Bu) increased (p

Ileal villus height (p

Colonic crypt depth was 10% greater (p = .012) in the 60Bu group compared with all other experimental groups at all time points (control = 225 ± 6.46, SCFA = 223 ± 6.46, 9Bu = 225 ± 6.46, 60Bu = 248 ± 6.30 µm). Taken together, the results show that butyrate treatments increased crypt-villus architecture, with the most marked effects on villus height. Butyrate induced these effects at acute time points only in the proximal intestine, whereas the effects in the distal intestine were sustained over the entire experimental period.

Intestinal and Epithelial Proliferation

Effect of intestinal cell proliferation was measured by Western blot analysis of transmural protein abundance of PCNA and histologic quantification of PCNA positive crypt cells. Relative amounts of PCNA protein abundance was not altered in the duodenum; however, histologic quantification of the number of crypt cells positively stained for PCNA was increased (p

Regardless of time, jejunal protein abundance of PCNA was increased (p = .009) by 12%, 9%, and 14% compared with control in the SCFA, 9Bu, and 60Bu groups, respectively. Ileal PCNA protein abundance was increased (p = .002) by 19%, 28%, and 32% compared with control in the SCFA, 9Bu, and 60Bu groups, irrespective of time. At each time point studied, the number of crypt cells positively stained for PCNA were increased in the jejunum (p

Regardless of time, colonie protein abundance of PCNA was increased (p = .004) in the SCFA, 9Bu, and 60Bu groups by 41%, 37%, and 49% compared with control, respectively. Histologie quantification of crypt cells positively stained for PCNA was increased (p

Intestinal Apoptotic Signaling Proteins

Apoptosis was measured in the intestine through quantification of proteins involved with the early regulation of the apoptotic signaling cascade. Bax (proapoptotic) was measured in the entire intestine, but because of segmental differences in the regulation of apoptosis by antiapoptotic proteins, Bcl-w was measured in the small intestine and Bcl-2 was measured in the colon. No effect of treatment was noted in the duodenum. Indicative of an antiapoptotic effect, the ratio of Bax:Bcl-w was decreased (p = .029) in the jejunum by the butyrate treatments (9Bu and 60Bu) compared with control, irrespective of time (Fig. 3A). In the ileum, all 3 supplemented TPN treatments (SCFA, 9Bu, and 60Bu) were decreased (p = .0001) compared with control, regardless of time (Fig. 3B). However, the antiapoptotic effect was less marked in the SCFA group than with the butyrate treatments (9Bu and 60Bu) in both the jejunum and ileum at all time points. Contrary to the small intestine, the colonie Bax:Bcl-2 ratio was increased (p

Plasma GLP-2 Concentration

Regardless of time, plasma GLP-2 concentration (pmol/L) was increased (p = .007) in the supplemented TPN treatments (SCFA, 9Bu, and 60Bu) compared with control (Fig. 4).

DISCUSSION

The trophic effect of SCFA-supplemented TPN on enhancing structural and functional intestinal adaptation after massive small bowel resection in an adult model is well established.20^23 However, their effects in the developing neonatal intestine, the specifie intestinotrophic SCFA, and the potential regulatory mechanism(s) are not well defined. Therefore, our study investigated the structural indices of intestinal adaptation using the neonatal piglet model, with supplementation of TPN containing a mixture of SCFA or butyrate alone over acute and chronic time points.

Our results indicate that supplementation of TPN with SCFA, or butyrate alone, can increase structural adaptation in the developing intestine. The butyrate treatments increased ileal DNA concentration per intestinal area, regardless of time, indicating mucosal hyperplasia us hypertrophy. However, no effect of supplemented TPN treatments on DNA concentration was noted in the duodenum and jejunum. This result likely occurred because DNA concentration was quantified in transmural homogenate and therefore was not as specific as if measured directly at the intestinal epithelium. Regardless, supplemented TPN treatments increased villus height in the duodenum, jejunum, and ileum as early as 4 hours post resection, and this response was maintained through 7 days in the jejunum and ileum. These results suggest that the effect of butyrate may be epithelial specific. To support this hypothesis, the increase in villus surface area in the duodenum, jejunum, and ileum likely resulted from increases in villus height because no change in midvillus width was noted in any intestinal segment. Thus, butyrate's primary effects appear to be on the villus epithelium, with secondary effects on the lamina propria. Additional support is provided by the result that the number of crypt cells positively stained with PCNA was increased by all 3 supplemented TPN treatments at all time points, in the jejunum and ileum, and that these epithelial-specific results were indeed more clear than those quantifying proliferative changes at the transmural level.

Examination of the results revealed that when significant increases in structural indices of intestinal adaptation occurred with the mixture of 3 SCFA, a significant effect was also present with butyrate; suggesting that the trophic effects noted with the mixture of the 3 SCFA can be stimulated by butyrate alone. This is supported by the report that 60 mmol/L butyrate administered colonically increased crypt cell proliferation rate within 2 days in the jejunum, whereas 100 mmol/L acetate and 20 mmol/L propionate had no effect in the jejunum.36 In addition, our results show that 9 mmol/L butyrate produces the same effect as 60 mmol/L butyrate in the neonatal intestine. Thus, the addition of butyrate above normal physiologic levels is not warranted as physiologic levels are sufficient to cause significant increases in structural indices of intestinal adaptation.

The process of adaptation is thought to reach a plateau at 1 to 2 years postresection.37,38 However, stimulators of adaptation can either facilitate a higher plateau (hyperadaptation) or reduce the time period to reach plateau (accelerated adaptation).39 Our data showed marked increases in structural indices of adaptation by systemic butyrate within 4 hours. Thus, TPN supplemented with butyrate appears to accelerate adaptation, thereby reducing the time to reach plateau, and facilitating a higher plateau by day 7 compared with control TPN. In addition, the timing of the treatment, whether it is early or late in the adaptation process, can also modulate the time period to reach plateau.39 In our study, butyrate was administered immediately after resection, but it is likely that butyrate can enhance intestinal adaptation and shorten the time period to full adaptation. Therefore, regardless of time postresection, the provision of SCFA before the initiation of enterai feedings may aid in the successful transition for children with SBS.

The crypt-villus remodeling induced by butyrate appears to be driven primarily by proliferation in the proximal intestine, whereas a more integrated response between proliferation and apoptosis was induced in distal segments. In the duodenum, supplemented TPN treatments had no effect on the regulation of apoptosis, although villus height was increased acutely and not chronically. This was likely the result of increases in proliferation or cell migration rate because increases in crypt cell PCNA were noted at 12 hours. In contrast to the duodenum, jejunal villus height was increased at all time points as a result of increased proliferation and decreased apoptosis, especially with the butyrate treatments. However, the ileum appears to be the intestinal segment most responsive to SCFA, as morphological-, cellular-, and epithelial-specific adaptations were increased, supporting the theory that the ileum has the greatest capacity for adaptation.11 Contrary to the small intestine, apoptosis was increased in the colon at 4 hours and 12 hours in the supplemented TPN treatments, but crypt cell proliferation was increased at 4 hours, 12 hours, 3 days, and 7 days. Thus, it is possible that within the colon, a new homeostatic set point is acutely established between proliferation and apoptosis during adaptation, as suggested by Stern and colleagues.

The acute effects in ileal crypt-villus architecture are probably caused by hyperplasia because DNA concentration was increased, along with indicators of cell proliferation, indicating an increase in the total number of cells, which could be caused by increased proliferation and possibly decreased cell turnover. However, the effect of SCFA on cell migration is poorly understood and not well studied. After intestinal resection, the remaining bowel increases rates of cell migration, resulting in shorter crypt and villus transit time.41,42 Even though cells migrate faster after resection, they have a greater distance to travel along the elongated villi; therefore, turnover time may be unchanged.11 Although migration was not examined in our study, according to the increased proliferation and decreased apoptosis results, it can be hypothesized that epithelial migration rate was increased at the acute time points. However, villus height did not continue to increase over time. Therefore, without direct measurements of migration, it is difficult to conclude that a new steady state was reached or if there was a change in cell migration or cell turnover at the chronic time points. It is possible that SCFA are directly responsible for the adaptive responses noted with SCFA-supplemented TPN. However, it is also likely that the adaptive responses noted with SCFA are mediated by GLP-2. Previous research has shown that SCFA increase the plasma concentration of GLP-2 and mRNA expression of proglucagon.21,23,26,28 Our results show that in neonatal piglets, TPN supplemented with SCFA (and more specifically butyrate), increase plasma GLP-2 concentrations regardless of time point examined. GLP-2 is an intestinal trophic hormone that has been shown to increase the structure and function of the small intestine. Thus, it may mediate the trophic effects of butyrate. Even though the primary stimulus for the distally located enteroendorine L-cells to secrete GLP-2 is enterai nutrition43,44 and even though plasma GLP-2 concentration is increased in resected animals compared with nonresected animals,45"47 our results support the hypothesis that systemic butyrate up-regulates the secretion of GLP-2. Our data indicate increases in GLP-2 secretion without luminal stimuli. Piglets receiving 80% jejunoileal resections supported receiving TPN supplemented with butyrate had increased levels of plasma GLP-2 compared with resected controls. Thus, SCFA increase plasma concentrations of GLP-2 possibly by affecting the secretion or expression of the hormone. According to previous and current results, the relationship between systemic butyrate and GLP-2 can only be described as an association that requires further research to determine a causal relationship.

There is much debate regarding the onset of structural and functional adaptation: whether one occurs before the other, which trophic factors act upon, and how the 2 are related in their mechanisms. Our results show that SCFA acutely increase structural indices of intestinal adaptation. Nonetheless, the question remains whether the newly proliferated cells have differentiated into functionally mature cells that would be capable of increasing intestinal absorption and digestion. It is thought that most structural adaptation takes place within 1 to 2 weeks after resection48,49 and that functional adaptation takes place within a few months after resection.50-52 Hence, increases in structure would lead to increases in function. Our recent results using the same experimental model indicate that glucose, amino acid, and dipeptide transport are increased by systemic butyrate administration at both acute and chronic time points.53'54 In the neonatal intestine, it appears that SCFA increase the functional capacity of the intestine by increasing the number of absorptive cells and increasing the translocation of nutrient transporters within cells.55-57 Thus, increases in intestinal structure related to SCFA most likely enhance intestinal function, thereby improving the prognosis of children with SBS.

In summary, administration of TPN supplemented with SCFA, or butyrate alone, enhances structural indices of intestinal adaptation in the neonatal piglet after massive small bowel resection by increasing proliferation and decreasing apoptosis. Therefore, to answer the 4 questions originally investigated:

1. SCFA do indeed enhance intestinal adaptation in the neonatal intestine after resection, as evidenced by increases in crypt-villus architecture.

2. Butyrate is in fact the SCFA responsible for the increase in structural indices of adaptation because of, the almost identical effects of the 3 SCFA (36 mmol/L acetate, 15 mmol/L propionate, and 9 mmol/L butyrate) compared with 9 mmol/L butyrate alone.

3. Intestinal structure is increased at both acute and chronic time points by butyrate, especially in the residual ileum.

4. Butyrate administration increases GLP-2 plasma concentration, thereby revealing a potential regulatory mechanism.

Thus, supplementation of TPN with butyrate may benefit infants with SBS by maximizing their intestinal absorptive area, thereby enabling these infants to successfully transition to enterai feedings.

ACKNOWLEDGMENT

The study was supported by grant NIDDK ROl DK 57682 from the National Institutes of Health. We are grateful for the expert experimental assistance of Brian Chung, PhD.

Discussant

Tom Jaksic, MD, PhD

Children's Hospital, Boston

Comments

As a pediatrie surgeon and director of a short bowel program I am much impressed by this work. The authors accurately point out that more than 10,000 patients in the United States have short-bowel syndrome (SBS) and further 30% of these patients are dependent upon total parenteral nutrition (TPN) for protected periods of time. Unfortunately long-term TPN in neonates may result in TPN-associated cholestasis, liver failure and even death. Multivariate analysis in such children indicates that institution of enterai nutrition, optimizing the length of residual small bowel, and re-establishment of bowel continuity are the key components to limiting TPN associated liver disease and decreasing mortality. It thus follows that enhancing small intestinal structure may be salutary, as it would allow for the more rapid institution of enterai nutrition.

In this study it was hypothesized that butyrate supplementation of TPN had a beneficial effect upon the structural aspects of intestinal adaptation in a TPN-fed neonatal piglet model. The experimental protocol employs an impressive randomized block design involving 120 neonatal piglets receiving central intravenous TPN. Remarkably none of the experimental animals had any technical difficulties during the experiment. Bartholome et al found that SCFA do enhance the structural aspects of intestinal adaptation, the effects extend to the one week period of the study, butyrate alone may induce these changes, and that GLP-2 is a potential mechanism mediating the trophic effects of butyrate.

This is a very extensive and pertinent study that required considerable effort. It extends findings from adult animals to neonatal piglets, and adds to previous work that indicates that butyrate administration may increase plasma levels of GLP-2. Bartholome and colleagues are to be congratulated for completing this ambitious investigation.

Questions

1. Does the enhancement of intestinal structure observed translate into measurable improvements of intestinal function, such as improved absorption?

2. Do you think that the 20-30% increase in serum GLP-2 levels associated with butyrate administration accounted for all the beneficiai structural effects noted?

3. Could you speculate upon the mechanisms by which butyrate may increase GLP-2 synthesis?

Author's Reply

Thank you very much. The functional aspect of the study is a very pertinent question that we asked and included within the design of the study. Another member of our lab is actually looking at the functional data and has found that glucose transport is acutely increased in the proximal intestine. When he looked at the other segments of the intestine, he saw an increase over the entire study period. So there is a significant effect of butyrate on glucose transport. he also looked at the transport of amino acids and peptides, finding that regardless of intestinal segment, there was an increase in amino acid and peptide transports at both acute and chronic time points. We are currently looking at measuring the protein abundance of glucose, amino acid and peptide transporters; specifically looking at the significant effect we saw with peptide transport through PepTl.

To answer the second question of whether these effects are due solely to GLP-2 or a combined effect of butyrate and GLP-2, there is evidence to show that GLP-2 can cause these same effects but there is some speculation that butyrate itself can induce these effects. So I am not sure if it is all completely due to an upregulation of GLP-2 by butyrate or if butyrate may have some independent effects of its own. In a study examining glucose transport, we investigated the hypothesis that butyrate may be acting independent of GLP-2, as luminal provision of butyrate increases glucose transport within 15 minutes as measured in modified Ussing chambers.

Speculation on some of the mechanisms by which butyrate may upregulate GLP-2 is an exciting area, which we are currently exploring in the lab. We are investigating the production, secretion and degradation of GLP-2 as well as examining its receptor abundance. With regard to GLP-2 production (based on our preliminary data as well as on other supporting studies), butyrate does increase the mRNA abundance of proglucagon. We show that when we look at the secretion of the other peptides produced and localized within the L-cell (being PYY and GLP-I), that their plasma concentration is not increased with regard to treatment. Thus, it looks like there is a specific effect of butyrate on GLP-2. We have proposed looking at the number of L-cells to see if there is a change in their number as well as the activity of prohormone convertase 1/3 within the L-cell. Finally, we have looked at the degradation of GLP-2 by dipeptidyl peptidase IV and found no change in the activity of dipeptidyl peptidase IV across treatments. Concluding that the increase in plasma GLP-2 is most likely due to butyrate increasing the production of GLP-2; and where that happens we are not sure, so we can only describe that as an association.

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Anne L. Bartholome, RD*; David M. Albin, MS*; David H. Baker, PhD*[dagger]; Jens J. Holst, MD§; and Kelly A. Tappenden, PhD, RD*[double dagger]

From the * Division of Nutritional Sciences, [dagger] Department of Animal Sciences, and [double dagger] Department of Food Science and Human Nutrition, University of Illinois at Urbana-Champaign, Urbana, Illinois; and the § Department of Medical Physiology, The Panum Institute, University of Copenhagen, Copenhagen, Denmark

Received for publication March 12, 2004.

Accepted for publication April 6, 2004.

Correspondence: Kelly A. Tappenden, PhD, RD, 443 Bevier Hall, 905

South Goodwin Avenue, Department of Food Science and Human Nutrition, University of Illinois at Urbana-Champaign, Urbana, IL

61801. Electronic mail may be sent to tappende@uiuc.edu.

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