Morphological, kinetic, membrane biochemical and genetic aspects of intestinal enteroplasticity

Abstract

The process of intestinal adaptation (“enteroplasticity”) is complex and multifaceted. Although a number of trophic nutrients and non-nutritive factors have been identified in animal studies, successful, reproducible clinical trials in humans are awaited. Understanding mechanisms underlying this adaptive process may direct research toward strategies that maximize intestinal function and impart a true clinical benefit to patients with short bowel syndrome, or to persons in whom nutrient absorption needs to be maximized. In this review, we consider the morphological, kinetic and membrane biochemical aspects of enteroplasticity, focus on the importance of nutritional factors, provide an overview of the many hormones that may alter the adaptive process, and consider some of the possible molecular profiles. While most of the data is derived from rodent studies, wherever possible, the results of human studies of intestinal enteroplasticity are provided.

INTRODUCTION

The intestine has an inherent ability to adapt morphologically and functionally in response to internal and external environmental stimuli. This process is called intestinal adaptation, or enteroplasticity. In fact, intestinal adaptation may be considered as a paradigm of gene-environment interactions. The array of phenotypic adaptations includes modification of brush border membrane (BBM) fluidity and permeability, as well as up- or down-regulation of carrier-mediated transport. Intestinal adaptation occurs following loss of a major portion of the small intestine (short bowel syndrome, SBS), following chronic ingestion of ethanol, following sublethal doses of abdominal irradiation, in diabetes, with aging, and with fasting and malnutrition[1–3].

Following intestinal resection, morphological and functional changes occur depending upon the extent and site of bowel removed, as well as external factors such as the lipid content of the diet (reviewed in Thiessen et al[4]). The increase in nutrient absorption from this process of enteroplasticity following resection compensates for the loss of mucosal absorptive surface area and minimizes the malabsorption that could otherwise occur. Therefore, intestinal adaptation has important implications in survival potential and welfare of the host[5]. In contrast, the adaptive process may be deleterious: for example, in diabetes this process enhances sugar and lipid uptake, exacerbating prevailing hyperglycemia, hyperlipidemia and obesity[6].

MECHANISMS

Mechanisms of intestinal adaptation occur at a variety of levels: physiological, cellular and molecular. Signals of adaptation may relate to various hormone levels, transcription factors, ATP levels, or to changes in concentration of luminal solutes[3]. Signals for and mechanisms of the enteroplasticity process may be different for the jejunum and ileum, as well as in the intestinal crypt and villous tip, explaining site-specific alterations and differences between crypt and villous enterocytes[1,2].

Rodents are commonly used in well-characterized models for assessing the process of intestinal adaptation[7]. For example, following small bowel resection in the rat, the remnant intestinal mucosa undergoes compensatory alterations in an attempt to restore normal absorptive capacity[8]. Morphological and functional changes include increases in crypt depth and villous length, enterocyte proliferation, as well as increased electrolyte, glucose and amino acid uptake[7,8].

The adaptive process has been defined in terms of transport kinetics. Changes usually occur in the value of maximal transport rate (Vmax) rather than in Michaelis affinity (Km) constant of specific nutrient transporters (sugars and amino acids)[9,10]. Furthermore, there may be alterations in permeability coefficients of nutrients transported passively such as short-, medium- and long-chain fatty acids and cholesterol[1,2,11]. Increased Vmax results from either up-regulation of the total number of transporters per enterocyte, increased number of transporting mucosal cells, or increase in the intrinsic activity of the transporter[12,13]. Intestinal resection also selectively changes passive permeability properties of the BBM, as demonstrated by increased uptake of fatty acids, an increase that is not due simply to the changes in mucosal surface area or the effective resistance of the intestinal unstirred water layer (UWL)[14]. Indeed, this altered permeability is due to changes in the lipophilic properties of the BBM caused by variations in-lipid content of the BBM[15], which represents part of the adaptive process.

DIABETES AND INTESTINAL RESECTION AS EXAMPLES

Intestinal adaptation in the rodent model of chronic diabetes involves changes at the transcriptional as well as the post-transcriptional level, leading to increased Na⁺-coupled sugar absorption[16]. After inducing acute hyperglycemia in rats, there is rapid up-regulation of glucose transport across the (enterocyte) basolateral membrane (BLM)[17]. In this model, both the vascular as well as luminal glucose infusion causes an increase in glucose transport capacity across the BLM[18]. No significant increase in BLM cytochalasin B binding or in GLUT2 protein abundance was observed, suggesting that there may be a post-translational event that increases the number of GLUT2 proteins available for transport, such as the movement of GLUT2 to the BLM from a preformed pool within the enterocyte. Alternatively, intrinsic activity of the transporter may be altered in the absence of changes in transporter protein abundance. Changes in intrinsic activity of glucose transporters have been observed with hyperglycemia[19], diabetes[20], low luminal glucose concentrations[12], and following activation of mitogen-activated protein kinase (MAPK) and P13K[13].

Following extensive intestinal resection, there is hyperplasia of the remaining small intestine, which is often accompanied by enhanced uptake of nutrients[21]. Alterations in the cell kinetics that result in modification of nutrition status may be specific or non-specific. Non-specific mechanisms involve alterations that result in changes in intestinal mucosal mass and/or villous surface area, leading to modifications in uptake of all nutrients, including those that are absorbed passively[22]. Specific mechanisms involve up- or down-regulation of transporters responsible for uptake of nutrients, such as sugars or amino acids[1,2].

THE IMPORTANCE OF MORPHOLOGY

The observation that morphological modifications may accompany intestinal adaptation in the rodent small bowel resection model was first made by Dowling and Booth[21]. The remaining intestine after resection is hyperplastic, with greater villous height and crypt depth, leading to enhanced mucosal surface area. While increased nutrient absorption is observed, the morphological changes do not necessarily solely explain alterations in nutrient uptake. For example, 1 wk after 80% small bowel resection, the remaining intestine increased its mass to 50%-70% of its pre-resection level, yet uptake of glucose increased only to approximately 33% of the pre-resection level[8].

It is clear that dynamic morphological parameters of the intestine may also adapt. For instance, crypt cell production rates or enterocyte migration rates along the villi change in some situations of intestinal adaptation[23]. It is important that morphological alterations be considered when estimating kinetic parameters of absorption. Morphological modifications such as blunting of mucosal growth or mucosal hyperplasia after intestinal resection are observed when dexamethasone is given subcutaneously[24]. Both transporter kinetics and dynamic morphological parameters are altered in the adaptive process, and the influence of resection on nutrient uptake is due to integration of these processes. This may be due to altered cell kinetics changing the population of enterocytes along the villus, thereby leading to variations in the number of cells with transporters, or activity of the transporters[25,26].

OTHER EXAMPLES

Many models of intestinal adaptation have been described: glucose uptake has been found to be increased during pregnancy[27], and lactation[28], with ingestion of a high carbohydrate diet[29], hyperglycemia[30], with diabetes[15], high alcohol intake[31] and after intestinal resection[32]. Glucose uptake is decreased with aging[33], external abdominal radiation[34], and with use of total parenteral nutrition[35]. Most transporters are up-regulated by levels of dietary substrate levels, and yet toxic substances and essential amino acids have the opposite effect[7,35–37]. These examples illustrate the diversity and variability of this enteroplasticity process.

Increases in nutrient absorption have been docu-mented[38–40] in humans following intestinal resection. The role of morphological changes in this process, however, has not been conclusively demonstrated. Remnant small bowel lengthening and dilatation has been noted in patients with SBS, suggesting morphological mechanisms in human intestinal adaptation[41]. However, the morphological adaptation typical in rodent models[21,42] (hypertrophy or hyperplasia) is not necessarily observed in the human adaptive response[43,44]. Several studies have shown no increase in villous height or crypt depth among patients who underwent intestinal resection, compared to healthy controls[39,45]. With the existence of various anatomical, physiological and biochemical differences between the human and rodent gastrointestinal tracts[46], and a conspicuous lack of comparable human studies, the clinical adequacy of the rat as a model of intestinal adaptation remains to be determined. Accordingly, caution must be used when attempting to extrapolate findings from rodent studies to the human population. Is there a better model? The neonatal piglet has been used in short bowel studies[47–49], and has been used to determine the effects of insulin-like growth factor-1 (IGF-l) and dietary manipulations[48,50]. The degree to which the results obtained using this model reflect human findings has yet to be determined, and the rodent remains a popular model for studies of intestinal adaptation.

DIETARY REGULATION

The topic of dietary regulation of intestinal gene expression has been reviewed[29,51]. Dietary constituents provide continual environmental signals that elicit expression of a host of genes that influence intestinal adaptation[52]. Every day, enterocytes are exposed to different nutrients that vary according to the nutrient intake of the host. For this reason, the intestine must be able to adapt to variations in the dietary load and composition[29,53]. The intestine, like many other biological and engineered systems, is quantitatively matched to prevailing peak loads with modest reserve capacities. Indeed, physiological capacities are optimal and most economical if they ascribe to the adage “enough, but not too much”[53]. Therefore, intestinal enzymes and transporters are characterized by a “safety factor”, a parameter that represents the ratio of its capacity to the load placed on it[54]. The maintenance of this reserve capacity is biosynthetically costly, but is necessary given the unpredictable nature of dietary contents.

Let us consider the process of enteroplasticity and parenteral vs enteral nutrition, dietary lipids, carbohydrates, proteins and polyamines.

Parenteral vs enteral nutrition

In rodent models using total parenteral nutrition (TPN), small bowel atrophy is well characterized[55–57]. Not surprisingly, the presence of luminal nutrients also contributes greatly to enteroplasticity[58].

Dietary lipids

The dietary fat content influences the uptake of hexoses and lipids into rabbit jejunum following ileal resection[14]. In using a rat model of SBS, early feeding of a high-fat diet increased lipid absorptive capacity of the intestinal remnant[59]. A high-fat diet decreased mucosal mRNA levels of the lipid binding protein FAT/CD36, and decreased oleic acid uptake by isolated enterocytes. Mice that were chronically fed a diet enriched in sunflower oil had increased liver fatty acid binding protein (L-FABP) mRNA levels in the small intestine[60]. The effect was specific to this gene, as the intestinal fatty acid binding protein (I-FABP) was unaffected.

Not only the amount of fat, but also the type of dietary fat may influence intestinal function. Keelan et al[61] tested the hypothesis that intestinal morphology and uptake of nutrients after resection of the distal half of the small intestine of rats responds to alterations in dietary content of saturated (SFAs) and polyunsaturated (PUFAs) fatty acids. Adult female Sprague-Dawley rats were subjected to a sham operation or to surgical resection of the distal half of the small intestine. Animals were fed chow for 3 wk, then either chow or isocaloric semisynthetic SFA or PUFA diets for a further 2 wk. The in vitro jejunal uptake of glucose was twice as high in animals that had undergone resection and were fed SFAs than in those fed PUFAs. Perhaps SFAs are necessary in the diet to ensure that adequate adaptation takes place.

Thiesen and colleagues examined the effect of dietary lipids on lipid uptake in rats post-resection. Intestinal resection had no effect on mRNA expression of early response genes (ERGs), proglucagon or the ileal lipid binding protein (ILBP), but was associated with reduced jejunal mRNA for ornithine decarboxylase (ODC) and for L-FABP[62]. These resection-associated changes in gene expression were not linked with alterations in intestinal uptake of long-chain fatty acids or cholesterol. In animals undergoing intestinal resection and fed SFA, there was a reduction in jejunal proglucagon mRNA expression as compared to those animals fed chow or PUFA. ODC mRNA expression in the jejunum of resected animals was reduced. Thus, dietary lipids modify uptake of lipids in resected animals, and ODC and proglucagon may be involved in this adaptive response[63].

The way by which dietary lipids alter gene expression and consequently change membrane composition and/or nutrient transport may be through activation of peroxisome proliferator-activated receptors (PPARs), hepatic nuclear factor-4 (HNF-4), nuclear factor κB (NFKB), and sterol response element binding protein-lc (SREBP-1c)[52]. By binding to these transcriptional factors, dietary lipids affect the rate of transcription and consequently the protein synthesis of nutrient transporters[51,64]. PPARs belong to the super-family of receptors that include the glucocorticosteroid receptor[65]. When the locally acting glucocorticosteroid budesonide was administered concomitantly with an SFA diet, jejunal uptake of glucose was increased but ileal uptake of fructose was reduced[66].

It has been suggested that dietary lipids participate in signal transduction involving activation of second messengers, such as cAMP, Ca²⁺ and diacylglycerol, thereby changing the mRNA expression[67]. Studies with glycosphingolipid have revealed the importance of these lipids and their metabolites in signaling pathways via the tyrosine kinase-linked receptors. This is a signal system mediated by protein kinase C (PKC), MAPK, other kinases, as well as by cytosolic Ca²⁺ concentration[68]. Additional new signals involved in adaptive intestinal response 3 d after 50% intestinal resection have been identified by cDNA microarray analysis. These include proline-rich protein 2, involved in wound healing; glutathione reductase, a gene involved in intestinal apoptosis; NF-2 family members, also involved in apoptosis; etoposide-induced p53-mediated apoptosis; basic Kruppe-like factor, a transcription factor that activates the promoter for IGF-1; and prothymosin-α, involved in cell proliferation[69,70]. These observations of altered expression of signals are useful to generate hypotheses that can be tested in future studies to establish whether these signals represent a primary or a secondary event in enteroplasticity.

The glycospingolipid, phospholipid, cholesterol and fatty acid composition of plasma membranes may be modified in mammalian cells[71]. For example, Keelan et al[72] demonstrated that alterations in dietary fatty acid saturation influence intestinal BBM phospholipid fatty acid composition in rats. The investigators proposed that previously reported diet-associated changes in active and passive intestinal transport are due at least in part to these alterations in the fatty acid composition in BBM phospholipids. A diet enriched with SFA is associated with increases in the saturation of BBM phospholipid fatty acids, while a diet enriched with PUFA is associated with an increase in the unsaturation of BBM phospholipid fatty acids[1,2].

Meddings[73] compared in vivo membrane lipid permeability within the same intestinal region, under conditions where membrane physical properties were radically altered by feeding rats an inhibitor of cholesterol synthesis. Marked reductions in membrane fluidity were observed due to replacement of membrane cholesterol with its precursor, 7-dehydrocholesterol. Associated with these alterations was a pronounced reduction in membrane lipid permeability. Therefore, BBM membrane lipid permeability, in vivo, appears to be correlated with the physical properties of the bilayer.

The degree of fatty acid unsaturation or saturation, as well as the cholesterol and ganglioside/glycosphingolipid content, are factors that influence fluidity of the BBM[74,75]. Changes in fluidity of the BBM may alter passive permeation of molecules and nutrients through this barrier, as well as conformation of binding sites on transporter proteins such as SGLT1, GLUTS[71,76]. For example, alterations in BBM fluidity influence passive uptake of lipids, as well as carrier-mediated D-glucose uptake[76,77]. While enhancement of fluidity increases the uptake of lipids, fluidization of BBM from enterocytes located on the villous tip decreases uptake of D-glucose to levels seen in the BBM from enterocytes located in the crypts[78].

While altered membrane lipid composition may act in part by changing viscosity or fluidity of the BBM, it may also alter the microenvironment surrounding the transporter and thereby modify transporter activity. Two types of specialized microdomains in the BBM have been identified: lipid rafts and caveolae. These regions are important in signal transduction as well as lipid and protein trafficking[79–81]. Lipid rafts are enriched in SFAs, cholesterol and gangliosides[80–82].

Feeding rats a diet containing gangliosides increases jejunal glucose uptake[83]. Feeding them a ganglioside-rich diet increases ganglioside content and decreases cholesterol content in the intestinal mucosa, plasma, retina and brain[84]. Similar changes in lipid composition of intestinal microdomains, or lipid rafts, occur following ganglioside feeding[85]. Although SGLT1 has been localized to these microdomains in renal epithelial cells[86], it is not known if sugar transporters reside in intestinal BBM microdomains. If this is the case, local changes in membrane fatty acids may affect the activity of transporter by altering the configuration of the protein, potentially exposing or masking transporter binding sites and thereby modifying nutrient uptake. Gangliosides may also influence intestinal sugar transport via alterations on pro-inflammatory mediators, many of which are known to influence intestinal sugar transport[87–89]. For example, in rats challenged with lipopolysaccharide, ganglioside feeding reduced the production of intestinal platelet activating factor, PGE2 and LTB4, as well as plasma levels of IL-1β and TNF-α[90].

Dietary carbohydrates

Dietary carbohydrate may induce the intestinal adaptive response by increasing the abundance of hexose transporters to facilitate a higher rate of sugar absorption[9]. In a murine model, intestinal glucose uptake was directly correlated with dietary carbohydrate load[29,36,91]. The effect of dietary carbohydrate on nutrient transporter abundance has been reported in several animal models. For instance, abundance of SGLT-1 in BBM and GLUT2 in the BLM was elevated in animals fed a high carbohydrate diet; associated with this enhanced level of protein was an increase in glucose absorption[17,92,93]. The GLUT5 transporter abundance was also elevated with enhanced consumption of dietary fructose, leading to increased fructose uptake[77].

Initiation of dietary glucose-induced adaptive response occurs in the intestinal crypts, where transport capacities of nutrient transporters are programmed[91,36,37,93]. Utilizing a mouse model, diet from a high to a low carbohydrate regimen reduced the amount of glucose transporter, as estimated from density of phlorizin binding. Enterocytes may adapt to the high carbohydrate diet by increasing crypt cell turnover rate, enhancing enterocyte migration rate, as well as by reprogramming the capability of nutrient transporters in the crypts to accommodate to the requirement for higher monosaccharide transporters[93]. Alteration in the density of glucose transporters was first observed in the crypt cells, and over a 3-d period was subsequently seen in the villous tip cells. Thus, crypt enterocytes respond to the high carbohydrate diet by increasing glucose transportation density. These cells then migrate up the villus over the next 3 d, contributing to the adaptation process enhancing glucose uptake.

Animals fed a glucose-enriched diet have increased glucose uptake, resulting from up-regulation of both BBM and BLM glucose transporters[17,92,94]. During early development, precocious introduction of dietary fructose causes enhanced expression of fructose transporters and fructose transport, without changing glucose uptake[93]. The substrates glucose and fructose specifically up-regulate their own transporters, SGLT-1 and GLUT5. In contrast, increases in essential amino acids or other substances that are potentially toxic at high levels (such as iron, calcium or phosphorous) are associated with no changes, or even reductions, in transport[35,95].

In many cases, other nutrients may be equal, or even more potent, inducers of the transporter than the transporter’s specific substrate. For example, young animals fed a diet enriched with PUFA have a decline in glucose uptake, as compared to animals fed an SFA-enriched diet[66,96,97]. Similarly, Vine et al[98] studied the effect of varying fatty acids on the passive and active transport properties of rat jejunum and found that an SFA-enriched diet increased Na⁺-dependent glucose uptake when compared to a diet enriched with n6 PUFA. In contrast, in aged rats, glucose uptake is increased by PUFA and not by SFA[33].

Dietary fiber also modulates intestinal nutrient uptake. For example, in dogs, a diet enriched with fermentable fiber increases glucose uptake and GLUT2 transporter abundance[99]. In vitro studies, in which rat intestinal tissue was incubated with B-glucan isolated from barley or oats, show reductions in uptake of stearic and linoleic acids (Drozdowski et al, 2005, unpublished observations). Furthermore, many studies have investigated the effect of TPN supplemented with short-chain fatty acids, the products of fiber fermentation. Increases in glucose uptake, GLUT2 mRNA and protein, and intestinal morphology were observed in normal rats as well as in rats following intestinal resection[100–103].

Dietary proteins

Dietary proteins also have an impact on intestinal morphology and active amino acid transport[37,104]. Both in vitro[105] and in vivo[104] rat experiments have shown that a high-protein diet increases amino acid uptake in the jejunum. Alteration in the amount of dietary protein induces reversible adaptation of non-essential amino acid transport rate[106]. Feeding a high-protein diet to mice induces a 77%-81% increment in uptake of non-essential amino acids[35], yet only a 32%-61% increase for essential amino acids. A protein-deficient regimen reduces uptake of non-essential amino acids, such as aspartate and proline, and maintains or increases uptake for essential amino acids and alanine.

Glutamine is a key metabolic fuel for enterocytes, mediating cellular nucleic acid synthesis and proliferation. Parenterally fed rats demonstrate decreased atrophy of the intestinal mucosa following glutamine supple-mentation[106]. Glutamine administration also normalizes the reduced levels of intestinal adaptation in rats receiving TPN following intestinal resection[107]. It is noteworthy that some studies of oral glutamine supplementation in the rat have failed to document more than temporary mucosal proliferation[108].

Other amino acids may inhibit intestinal adaptation. Sukhotnik et al[109] examined effects of parenteral administration of the nitric oxide precursor arginine to rats following 75% small bowel resection. Arginine supplementation was associated with lower cell proliferation and greater enterocyte apoptosis. Thus, the nature of the adaptive response depends upon the type of amino acid and the needs of the animal.

Polyamines

Polyamines are found in all eukaryotic cells[110], and they play an important role in growth and differentiation[111]. Polyamines are obtained either from the diet, or via synthesis from ornithine[112]. Luminal perfusions of polyamines rapidly (in less than 5 min) enhance intestinal glucose uptake in rats and increase BBM SGLT-1 protein[113].

Polyamine synthesis or uptake may be an important event that initiates adaptive hyperplasia observed in the intestinal remnant after partial small bowel resection. Enteral diets supplemented with ornithine alpha-ketoglutarate (OKG), a precursor for arginine, glutamine and polyamines, enhances intestinal adaptation in models of intestinal resection[114,115]. Indeed, studies by both Tappenden et al[116] and Thiesen et al[62] suggest that ODC, a key enzyme in polyamine synthesis, may mediate the adaptive process in rats that is stimulated by the administration of either glucocorticosteroids or short-chain fatty acids to rats following intestinal resection.

HORMONAL REGULATION

Glucocorticosteroids (GCs)

Numerous hormones modify the form and function of the intestine. It is not clear whether any of the dietary features that modify the intestinal adaptive process do so by way of hormonal alterations. In a model of extensive intestinal resection (50% enterectomy), the remaining proximal and distal intestinal remnants were adequate to assess the morphology and function at these sites[9,106]. The GC prednisone had no effect on intestinal uptake of glucose or fructose in these resected animals[62]. In contrast, the locally acting steroid budesonide increased by over 120% the value of the jejunal Vmax for the uptake of glucose, and increased by over 150% the ileal uptake of fructose. The protein abundance and mRNA expression of SGLT-1, GLUTS, GLUT2 and Na⁺/K⁺ APTase α1 and β1 did not explain this enhancing effect of budesonide on glucose and fructose uptake. Budesonide, prednisone and dexamethasone reduced jejunal expression of the early response gene c-jun. In resected animals, the abundance of the mRNA of ODC in the jejunum was reduced, and GCs reduced jejunal expression of mRNA for proglucagon. These data suggest that enhancing influence of GC on sugar uptake in resected animals may be achieved by post-translational processes involving signalling with c-jun, ODC and proglucagon, or other as yet unknown signals.

In contrast, the uptake of D-fructose by GLUT5 was similarly increased with budesonide and with prednisone. Increases in the uptake of fructose was not due to variations in weight of intestinal mucosa, food intake, or in GLUT5 protein or mRNA expression. There were no steroid-associated changes in mRNA expression of c-myc, c jun, c-fos, of proglucagon, or of selected cytokines. However, the abundance of ileal ODC mRNA was increased with prednisone. Giving post-weaning rats budesonide or prednisone in 4-wk doses equivalent to those used in clinical practice increases fructose but not glucose uptake. This enhanced uptake of fructose was likely regulated by post-translational processes[62].

Growth hormone (GH)

GH has been suggested as possessing pro-adaptive properties[117]. In rats and piglets, GH administration results in an increase in small bowel length and function per unit length[118]. Hypophysectomized rats undergo mucosal hypoplasia of the small bowel, as well as a reduced adaptive response following resection that is restored by GH[119]. In contrast, transgenic mice expressing elevated levels of GH experience hypertrophy of the small intestine[118]. IGF-1 expression in the small bowel is regulated by GH and is believed to induce enterotrophic effects following resection[120,121]. In a rat model of SBS, acute IGF-1 treatment of TPN-fed rats produced sustained jejunal hyperplasia, and facilitated weaning from parenteral to enteral nutrition[122]. GH administration to normal rats has been reported to have positive effects on mucosal growth and intestinal adaptation following massive resection[123], although contradictory data exist[124,125]. Human and rabbit studies have indicated that increased nutrient transporter activity devoid of morphological changes may be the method of GH-induced intestinal adaptation[126].

GH administration inhibits liberation of glutamine from muscle during catabolic states in humans[127]. This suggests a possible role for combining GH and glutamine to achieve optimal adaptation. Trials investigating any such synergism in the rat have yielded conflicting results. Some studies have failed to demonstrate an additive effect of GH and glutamine in the enhancement of post-resection intestinal adaptation[128], while others have documented a positive synergistic effect[129]. For example, GH has been shown to enhance the absorption of amino acids using ex vivo human BBM vesicles[129]. An intestinal mucosal GH receptor has been described in rats and humans[130], and GH promotes cell differentiation and clonal expansion of these differentiated cells[131].

Human studies have suggested that the efficacy of GH and/or glutamine therapy in the adaptive response of the small bowel may be based heavily upon the clinical status of the patient[132]. Evaluation of the effect of such may facilitate further understanding of the pathology and physiology of the bowel adaptation process, as well as more clearly defining positive predictive indicators of the bowel’s ability to be rehabilitated. Existing human data indicate that administration of high concentrations of GH can actually increase patient morbidity and mortality[133].

In studies of home parenteral nutrition (HPN)-dependent patients with SBS, the use of high-dose recombinant human GH (0.4 mg/kg per day) in controlled[133,134] and uncontrolled studies[135] has led to variable results. Patients were given glutamine supplements by mouth or parenterally, and their diet was modified. In the randomized, placebo-controlled study of Scolapio et al[133], subjects ingested a standardized 1500 kcal/d diet, which is clearly different from the hyperphagic diet consumed by many SBS patients[136], and which may contribute to the physiological adaptation that occurs in the remaining intestine after extensive resection. It is unclear whether glutamine is beneficial for the adaptive response in humans. In rat models of SBS, it is unclear whether glutamine supplementation is efficacious for the adaptive process[137,138]. Furthermore, both a hyperphagic diet and absence of malnutrition are needed for humans to achieve optimal intestinal adaptation[41,139].

When HPN-dependent patients with SBS were provided a usual ad libitum hyperphagic diet, and given low doses of GH (0.05 mg/kg per day) for 3 wk, there was significant improvement in intestinal absorption of energy (15% ± 5%), nitrogen (14% ± 6%) and carbohydrate (10% ± 4%)[140]. Increased food absorption represented 37% ± 16% of total parenteral energy delivery. Body weight, lean body mass, D-xylose absorption, IGF-1, and IGF binding protein 3 increased, whereas uptake of GH binding protein decreased. During treatment with GH, improvement in net intestinal absorption compared with placebo was 427 ± 87 kcal/d, representing 19% ± 8% of the total energy expenditure required to obtain energy balance equilibrium in patients with SBS[136].

From a review of literature in this area, Matarese et al[140] noted that there were differences in gastrointestinal (GI) anatomy, dietary compliance, nutritional status, presence of mucosal disease, and diagnosis both within and between studies. These authors concluded that “administering recombinant human growth hormone alone or together with glutamine with or without a modified diet may be of benefit when the appropriate patients are selected for treatment”.

IGF-1

IGF-1 proved to be efficient in increasing intestinal adaptation following resection in rats. IGF-l treatment following 70% jejuno-ileal resection attenuated fat and amino acid malabsorption[141] and increased total gut weight by up to 21%. The IGF-1 receptor was increased in the jejunum and colon due to resection. Resection also increased circulating IGF-binding proteins (IGFBPs). IGF-1 treatment had no effect on IGF-1 mRNA or IGF-1 receptor density, but increased IGFBP-5 in the jejunum[142]. This increase in IGFBP-5 was correlated with jejunal growth after IGF-1 treatment[142].

IGF-1 treatment in resected rats significantly increased jejunal mucosal mass by 20% and mucosal concentrations of protein and DNA by 36 and 33%, respectively, above the response to resection alone[143]. These changes reflected an increase in enterocyte proliferation and an expansion of the proliferative compartment in the crypt. No further decrease in enterocyte apoptosis, or increase in enterocyte migration[144].

IGF-1 treatment may also facilitate weaning from parenteral to enteral nutrition. After 60% jejunoileal resection plus cecectomy, rats treated with recombinant human IGF-1 (3 mg/kg body weight per day) or control vehicle were maintained exclusively with TPN for 4 d, and were then transitioned to oral feeding. TPN and IGF-1 were stopped 7 d after resection, and rats were maintained with oral feeding for 10 more days. Acute IGF-1 treatment induced sustained jejunal hyperplasia, as suggested from the presence of greater concentrations of both jejunal mucosal protein and DNA, and was associated with maintenance of a greater body weight and serum IGF-1 concentrations[122].

Using male transgenic mice with targeted smooth muscle IGF-1 over-expression[145], these as well as non-transgenic littermates underwent 50% proximal small bowel resection. Growth factor over-expression led to a unique mucosal response characterized by a persistent increase in remnant intestinal length and an increase in mucosal surface area. Therefore, IGF-1 signaling from within the muscle layer may be important in resection-induced intestinal adaptation. In summary, IGF-1 shows promise as a hormone which may prove to be of clinical significance in nutritional regulation and the modification of intestinal absorption in the short and long term[138].

Epidermal growth factor (EGF)

EGF up-regulates intestinal nutrient transport[146]. This effect is mediated by PKC and P13K[147], and involves redistribution of SGLT-1 from microsomal pools to the BBM[148,149]. After massive intestinal resection, endogenous EGF is increased in saliva and is decreased in urine[150]. EGF stimulates intestinal adaptation after intestinal resection: the BBM surface area and total absorptive area increased until day 10, and EGF treatment induced a further increase in BBM surface area[151]. In a study by O’Brien and colleagues[152], mice underwent 50% small bowel resection or sham operation, and were then given orally an EGF receptor (EGFR) inhibitor (ZD1839, 50 mg/kg per day) or control vehicle for 3 d. ZD1839 prevented EGFR activation, as well as the normal postresection increases in ileal wet weight, villus height, and crypt depth. Enterocyte proliferation was reduced two-fold in the resection group by ZD1839. These results more directly confirm the requirement of a functional EGFR as a mediator of postresection adaptation response. Previous work has demonstrated that the EGFR is predominantly located on the BLM of enterocytes[153], but after small bowel resection the EGFR shows redistribution from the BLM to the BBM, with no change in the total amount of EGFR[154]. It is not known how this redistribution occurs. This is an important point, since modification of this process may represent a useful means to accelerate the intestinal adaptive process.

Laser capture microdissection (LCM) microscopy was used to elucidate the specific cellular compartment(s) responsible for postresection changes in EGFR expression[155]. Mice underwent a 50% proximal resection or sham operation, and after 3 d, frozen sections were taken from the remnant ileum. Individual cells from the villi, crypt, muscularis and mesenchymal compartments were isolated. EGFR mRNA expression for each cell compartment was quantified using real-time reverse transcriptase polymerase chain reaction (RT-PCR). EGFR expression was increased two-fold in the crypts after resection. This supports the hypothesis that EGFR signaling is crucial for the mitogenic stimulus for adaptation. The additional finding of increased EGFR expression in the muscular compartment is novel and may imply a role for EGFR in the muscular hyperplasia seen after massive small bowel resection. As noted previously, it is of interest that the muscle layer also appears to play a role in the adaptive response to IGF-1[155].

Treatment of resected rats with EGF has been studied: male juvenile rats underwent either transection or ileocecal resection leaving a 20-cm jejunal remnant[156]. Resected animals were treated orally with placebo or recombinant human EGF. Resected EGF-treated animals lost significantly less weight than those in the transection group, absorbed significantly more 3-0 methylglucose, and had reduced intestinal permeability as determined by the lactulose/mannitol ratio. Work by Chung et al[149] using rabbits showed that intestinal resection altered SGLT-1 mRNA and protein expression along the crypt-villous axis, with expression being highest in the mid-villous region. Oral EGF normalized SGLT-1 expression, resulting in a gradient of increasing expression from the base of the villous to the villous tip.

Nakai and colleagues[157] investigated the role of EGF in stimulating intestinal adaptation following small bowel transplantation. Treatment of rats with EGF (intraperitoneally for 3 d) following intestinal transplantation resulted in increased glucose absorption, SGLT-1 abundance and villous height and crypt depth in the graft. Clearly, there are sufficient animal data to support studies of the potential pro-adaptive role of EGF in humans.

Keratinocyte growth factor (KGF)

In a study by Yang et al[158], adult C57BL/6J mice were randomized to 55% mid-small bowel resection, resection with KGF administration, or a sham-operated (control) group, and were killed at day 7. Ussing chamber studies showed that KGF increased net transepithelial absorption of 3-0-methyl glucose as well as sodium-coupled alanine absorption, but had no effect on epithelial permeability barrier function. Epithelial cells were separated along the crypt-villous axis with LCM, and epithelial KGF receptor (KGFR) mRNA abundance was studied using real time RT-PCR. KGF up-regulated KGFR mRNA abundance, predominately in the crypt and the lower portion of the villus.

Leptin and ghrelin

Leptin plays an important role in the regulation of body fat and satiety (reviewed in Jequier[159]). Leptin reduces food intake[160] and leptin-deficient mice develop obesity[161]. Leptin may be a potential growth factor for the normal rat small intestine. The effect of 14 d of parenteral leptin administration (2 μg/kg per day) to rats following 80% small bowel resection was studied. Leptin was associated with a 44% increase in galactose absorption and a 14% increase in GLUT-5 abundance, but with no change in DNA content or in SGLT abundance. These findings suggest that leptin may potentially be clinically useful in patients with impaired intestinal function[162].

Ghrelin is a gastric hormone that is released in response to enteral nutrients. It has an opposite effect when compared to leptin, as it stimulates food intake[163]. The role of ghrelin in intestinal adaptation is unknown.

Glucagon-like peptide 2 (GLP-2)

Animal studies have demonstrated a potential role for GLP-2 in the adaptive response following intestinal resection[143]. Plasma GLP-2 levels rise following intestinal resection in rats[164–166]. In a study by Dahly et al[143], rats were subjected to 70% mid-jejunoileal resection or ileal transection, and were maintained with TPN or oral feeding. Resection-induced adaptive growth in TPN- and orally-fed rats was associated with a significant positive correlation between increases in plasma bioactive GLP-2 and proglucagon mRNA abundance in the colon of TPN-fed rats and in the ileum of orally fed rats. While these increases were transient in the TPN-fed group, luminal nutrients produced a sustained increase detected at 3 and 7 d post-resection. These data support a significant role for endogenous GLP-2 in the adaptive response to mid-small bowel resection in both TPN and orally fed rats[167].

Correlations between post-resection GLP-2 levels, morphological indices, crypt cell proliferation rates and nutrient absorption have been made[168]: an inverse correlation was found between post-prandial GLP-2 levels and fat or protein absorption, as assessed by a 48-h balance study. These results, along with data obtained from studies showing that GLP-2 immunoneutralization inhibits post-resection adaptation[169], further implicate GLP-2 as a post-resection mediator of adaptation.

GLP-2 administration in rats increases the adaptive response to massive intestinal resection[170]. In this study, Sprague-Dawley rats were divided into two groups, one with a 75% mid-jejunum-ileum resection, and a second sham operated group. Animals were treated with 0.1 pg/g GLP-2 analog (protease resistant human GLP-2) or placebo given subcutaneously twice daily for 21 d. The groups were compared measuring the total weight of the rats, and mucosal mass per centimeter. Administration of GLP-2 or its analogs was associated with an increase of the mucosal mass in the proximal jejunum and terminal ileum. While resection reduced D-xylose excretion, GLP-2 restored D-xylose excretion to levels above control values within 21 d of administration. This indicates that GLP-2 has a positive effect on intestinal morphology and absorptive function following resection.

Martin et al[170] investigated the effects of GLP-2 in a TPN-supported model of experimental SBS. Juvenile Sprague-Dawley rats underwent a 90% small intestinal resection and were randomized to three groups: enteral diet and intravenous saline infusion, TPN only, or TPN + 10 μg/kg per h GLP-2. TPN plus GLP-2 treatment resulted in increased bowel and body weight, villous height, intestinal mucosal surface area, and crypt cell proliferation. GLP-2 reduced the lactulose-mannitol ratio, indicating that GLP-2 lowered intestinal permeability when compared with the TPN alone. GLP-2 increased serum GLP-2 levels as well as intestinal SGLT-1 protein abundance compared with either TPN or enteral groups. This study demonstrates that GLP-2 is capable of stimulating intestinal adaptation in the absence of enteral feeding.

Since a number of hormones and growth factors have been shown to influence intestinal function, Washizawa et al[171] compared the effects of GLP-2, GH and KGF on markers of gut adaptation following massive small bowel resection. KGF increased goblet cell numbers and TTF3, a cytoprotective trefoil peptide, in the small bowel and the colon. While both GH and KGF increased colonic mucosal growth, GLP-2 exerted superior trophic effects on jejunal growth. GLP-2 also increased the glutathione/glutathione disulfide ratio, resulting in improved mucosal glutathione redox status throughout the bowel. Because of the differential effects of GLP-2, GH and KGF on gut adaptation following massive small bowel resection, the authors conclude that a combination of these agents may be most beneficial.

A pilot study to determine efficacy of GLP-2 in patients with SBS has been completed. A non-placebo controlled study was conducted in eight patients with SBS with an end-enterostomy type of anastomosis (six had Crohn’s disease and four were not receiving HPN)[172]. Treatment with GLP-2 (400 μg subcutaneously twice a day for 35 d) increased intestinal absorption of energy, body weight, and lean body mass. Crypt depth and villous height were also increased in five and six patients, respectively.

A review by Jeppesen[173] on the role of GLP-2 in treatment of SBS concludes that: “Currently, hormonal therapy in short-bowel patients should be considered experimental and it is only recommended in research studies”[11]. The optimal duration and concentration requirements for GLP-2 to induce beneficial effects on intestinal secretion, motility, morphology and most importantly absorption, are not known. Optimal dosage and administration of this new treatment to short-bowel patients may eventually result in long-term improvements in nutritional status and independence of parenteral nutrition in a larger fraction of short-bowel patients.

OTHER POSSIBLE SIGNALS OF INTESTINAL ADAPTATION

Dodson et al[174] identified three subsets of genes that were up-regulated by constructing a cDNA library from the remnant ileum of resected rats. This library was screened, and subtractive hybridization was used to identify genes that were induced following resection. These included genes involved with regulating the absorption and metabolism of nutrients. For example, L-FABP, apolipoprotein A-IV, cellular retinal binding protein II and ileal lipid binding protein were identified as genes that were induced following 70% intestinal resection in rats. Genes involved in cell cycle regulation were also identified. For example, phosphorylation and dephosphorylation are important regulators of the cell cycle, and PP1S, a subunit of a serine/threonine phosphatase was indeed up-regulated. Grp78, a member of the heat shock protein family was also increased. Grp78 resides in the endoplasmic reticulum and acts as a chaperone during protein assembly and transport. It may also have a protective role, and prevent apoptosis as a way of promoting the proliferative response following intestinal resection[175,176].

Rubin et al[177] further characterized the molecular and cellular mechanisms following 70% resection in rats. An immediate early gene, PC4/TIS7, was markedly increased soon after resection, with levels returning back to normal by 1 wk post-resection. Although the function of this protein is unknown, it may be related to cytodifferentiation as it is expressed only in the villus and not in the crypts.

Erwin et al[70] used cDNA microarrays to gain insight into the mechanism of intestinal adaptation. Mice underwent a 50% intestinal resection, and 3 d afterwards RNA was extracted from the remnant ileum. Multiple genes were induced, and fall into four categories: (1) apoptosis, DNA synthesis, repair and recombination (ten genes); (2) oncogenes, tumor suppressors, cell cycle regulators (three genes); (3) stress response, ion channels and transport (four genes); (4) transcription factors and general DNA-binding proteins (one gene).

Many of the genes (ODC, c-neu, glucose-related protein, IGFBP-4) that were identified agreed with the results of other studies of intestinal resection. For example, ODC was increased in this study, and this agrees with previous findings that showed ODC to be involved in the adaptive process[60,116,178]. Some new factors were also identified including glutathione reductase (involved in apoptosis), basic Kruppel-like factor (transcriptional regulator that activates the IGF promoter), prothymosin-α (associated with increased cell proliferation), and eteposide-induced p53-responsive mRNA (stress response protein involved in p53 mediated apoptosis).

Stern et al[69] performed a similar analysis of gene expression following 50% intestinal resection in rats. The gene with the largest increase was identified as Sprr2, a novel gene not previously known to be involved in intestinal adaptation. EGF administration post-resection further increased sprr2 expression, and enhanced the adaptive response. This protein plays a role in terminal differentiation of stratified squamous epithelium. Its role in the intestinal epithelium is unclear and warrants further investigation.

Finally, a variety of other signals have been described as possibly playing a role in the process of intestinal adaptation. These include prostanoids[179], uncoupling proteins[180], PPAR-α, transforming growth factor-α[181], SPARC (secreted protein, acidic and rich in cysteine[182], Bcl-2[183], endothelin-1[184], erythropoietin[185], the GATA family of zinc finger transcription factors[186], hepatocyte growth factor[187], the ERGs[188], PC4/TIS7[177] and epimorphin[189]. Augmented Wnt signaling has been shown to enhance the adaptive response to massive small bowel resection[190]. Several of these signals may be useful to modify in a clinical setting to enhance the intestinal adaptive response.

Microarray technology is a powerful tool that is constantly developing into a more sophisticated technique of identifying novel genes involved in physiological processes. Intestinal adaptation awaits further characterization by hypothesis-testing studies. From the information that is available at this time, it is clear that genes regulating the cell cycle, proliferation, differentiation and apoptosis are important components of the adaptive process, leading to enteroplasticity.