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The unfolded protein response (UPR) is a complex adaptive signaling pathway activated by the accumulation of misfolded proteins in the endoplasmic reticulum (ER). ER stress (ERS) triggers a cascade of responses that converge upon C/EBP homologous protein (CHOP) to drive inflammation and apoptosis. Herein, we sought to determine whether liver injury and fibrosis after small bowel resection (SBR) were mediated by a maladaptive hepatic ERS/UPR. C57BL/6 mice underwent 50% proximal SBR or sham operation. Markers of liver injury and UPR/ERS pathways were analyzed. These were compared with experimental groups including dietary fat manipulation, tauroursodeoxycholic acid (TUDCA) treatment, distal SBR, and global CHOP knockout (KO). At 10 wk, proximal SBR had elevated alanine aminotransferase/aspartate aminotransferase (ALT/AST) (P < 0.005) and greater hepatic tumor necrosis factor-α (TNFα) (P = 0.001) and collagen type 1 α1 (COL1A1) (P = 0.02) than shams. SBR livers had increased CHOP and p-eIF2α, but were absent in activating transcription factor 4 (ATF4) protein expression. Low-fat diet (LFD), TUDCA, and distal SBR groups had decreased liver enzymes, inflammation, and fibrosis (P < 0.05). Importantly, they demonstrated reversal of hepatic UPR with diminished CHOP and robust ATF4 signal. CHOP KO-SBR had decreased ALT but not AST compared with wild-type (WT)-SBR (P = 0.01, P = 0.12). There were no differences in TNFa and COL1A1 (P = 0.09, P = 0.50). SBR-induced liver injury, fibrosis is associated with a novel hepatic UPR/ERS response characterized by increased CHOP and decreased ATF4. LFD, TUDCA, and ileocecal resection rescued the hepatic phenotype and reversed the UPR pattern. Global CHOP KO only partially attenuated liver injury. This underscores the significance of disruptions to the gut/liver axis after SBR and potentiates targets to mitigate the progression of intestinal failure-associated liver disease. [ABSTRACT FROM AUTHOR]

Loss of functional small bowel surface area causes short bowel syndrome (SBS), intestinal failure, and parenteral nutrition (PN) dependence. The gut adaptive response following resection may be difficult to predict, and it may take up to 2 yr to determine which patients will wean from PN. Here, we examined features of gut microbiota and bile acid (BA) metabolism in determining adaptation and ability to wean from PN. Stool and sera were collected from healthy controls and from patients with SBS (n = 52) with ileostomy, jejunostomy, ileocolonic, and jejunocolonic anastomoses fed with PN plus enteral nutrition or who were exclusively enterally fed. We undertook 16S rRNA gene sequencing, BA profiling, and 7α-hydroxy-4-cholesten-3-one (C4) quantitation with LC-MS/MS and serum amino acid analyses. Patients with SBS exhibited altered gut microbiota with reduced gut microbial diversity compared with healthy controls. We observed differences in the microbiomes of patients with SBS with ileostomy versus jejunostomy, jejunocolonic versus ileocolonic anastomoses, and PN dependence compared with those who weaned from PN. Stool and serum BA composition and C4 concentrations were also altered in patients with SBS, reflecting adaptive changes in enterohepatic BA cycling. Stools from patients who were weaned from PN were enriched in secondary BAs including deoxycholic acid and lithocholic aicd. Shifts in gut microbiota and BA metabolites may generate a favorable luminal environment in select patients with SBS, promoting the ability to wean from PN. Proadaptive microbial species and select BA may provide novel targets for patient-specific therapies for SBS. NEW & NOTEWORTHY Loss of intestinal surface area causes short bowel syndrome, intestinal failure, and parenteral nutrition dependence. We analyzed the gut microbiota and bile acid metabolome of a large cohort of short bowel syndrome adult patients with different postsurgical anatomies. We report a novel analysis of the microbiome of patients with ileostomy and jejunostomy. Enrichment of specific microbial and bile acid species may be associated with the ability to wean from parenteral nutrition. [ABSTRACT FROM AUTHOR]

Intestinal failure-associated liver disease is a major morbidity associated with short bowel syndrome. We sought to determine if the obesity-resistant mouse strain (129S1/SvImJ) conferred protection from liver injury after small bowel resection (SBR). Using a parenteral nutrition-independent model of resection-associated liver injury, C57BL/6J and 129S1/SvImJ mice underwent a 50% proximal SBR or sham operation. At postoperative week 10, hepatic steatosis, fibrosis, and cholestasis were assessed. Hepatic and systemic inflammatory pathways were evaluated using oxidative markers and abundance of tissue macrophages. Potential mechanisms of endotoxin resistance were also explored. Serum lipid levels were elevated in all mouse lines. Hepatic triglyceride levels were no different between mouse strains, but there was an increased accumulation of free fatty acids in the C57BL/6J mice. Histological and serum markers of hepatic fibrosis, steatosis, and cholestasis were significantly elevated in resected C57BL/6J SBR mice as well as oxidative stress markers and macrophage recruitment in both the liver and visceral white fat in C57BL/6J mice compared with sham controls and the 129S1/SvImJ mouse line. Serum endotoxin levels were significantly elevated in C57BL/6J mice with significant elevation of hepatic TLR4 and reduction in PPARα expression levels. Despite high levels of serum lipids, 129S1/SvImJ mice did not develop liver inflammation, fibrosis, or cholestasis after SBR, unlike C57BL/6J mice. These data suggest that the accumulation of hepatic free fatty acids as well as increased endotoxin-driven inflammatory pathways through PPARα and TLR4 contribute to the liver injury seen in C57BL/6J mice with short bowel syndrome. NEW & NOTEWORTHY Unlike C57BL/6 mice, the 129S1/SvImJ strain is resistant to liver inflammation and injury after small bowel resection. These disparate outcomes are likely due to the accumulation of hepatic free fatty acids as well as increased endotoxin-driven inflammatory pathways through PPARα and TLR4 in C57BL/6 mice with short bowel syndrome. [ABSTRACT FROM AUTHOR]

After 50% proximal small bowel resection (SBR) in mice, we have demonstrated hepatic steatosis, impaired glucose metabolism without insulin resistance, and increased pancreatic islet area. We sought to determine the consequences of SBR on pancreatic β-cell morphology, proliferation, and expression of a key regulatory hormone, glucagon-like peptide-1 (GLP-1). C57BL/6 mice underwent 50% SBR or sham operation. At 10 wk, pancreatic insulin content and secretion was measured by ELISA. Immunohistochemistry was performed to determine structural alterations in pancreatic α-and β-cells. Western blot analysis was used to measure GLP-1R expression, and immunoassay was used to measure plasma insulin and GLP-1. Experiments were repeated by administering a GLP-1 agonist (exendin-4) to a cohort of mice following SBR. After SBR, there was pancreatic islet hypertrophy and impaired glucose tolerance. The proportion of α and β cells was not grossly altered. Whole pancreas and pancreatic islet insulin content was not significantly different; however, SBR mice demonstrated decreased insulin secretion in both static incubation and islet perfusion experiments. The expression of pancreatic GLP-1R was decreased approximately twofold after SBR, compared with sham and serum GLP-1, was decreased. These metabolic derangements were mitigated after administration of the GLP-1 agonist. Following massive SBR, there is significant hypertrophy of pancreatic islet cells with morphologically intact α- and β-cells. Significantly reduced pancreatic insulin release in both static and dynamic conditions demonstrate a perturbed second phase of insulin secretion. GLP-1 is a key mediator of this amplification pathway. Decreased expression of serum GLP-1 and pancreatic GLP-1R in face of no change in insulin content presents a novel pathway for enteropancreatic glucose regulation following SBR. NEW & NOTEWORTHY Metabolic changes occur following intestinal resection; however, the effects on pancreatic function are unknown. Prior studies have demonstrated that glucagon-like protein-1 (GLP-1) signaling is a crucial player in the improved insulin sensitivity after bariatric surgery. In this study, we explore the effect of massive small bowel resection on gut hormone physiology and provide novel insights into the enteropancreatic axis. [ABSTRACT FROM AUTHOR]

Effective therapies are limited for patients with parenteral nutritiondependent short bowel syndrome. We previously showed that intestinal expression of the transcriptional coregulator tetradecanoyl phorbol acetate-induced sequence 7 (tis7) is markedly increased during the adaptive response following massive small bowel resection and tis7 plays a role in normal gut lipid metabolism. Here, we further explore the functional implications of tis7 deletion in intestinal lipid metabolism and the adaptive response following small bowel resection. Intestinal tis7 transgenic (tis7tg), tis7-/-, and wild-type (WT) littermates were subjected to 50% small bowel resection. Mice were fed a control or a high-saturated-fat (42% energy) diet for 21 days. Survival, body weight recovery, lipid absorption, mucosal lipid analysis, and the morphometric adaptive response were analyzed. Quantitative real-time PCR was performed to identify tis7 downstream gene targets. Postresection survival was markedly reduced in high-fat, but not control, diet-fed tis7-/- mice. Decreased survival was associated with anastomotic inflammation and intestinal obstruction postresection. High-fat, but not control, diet-fed tis7-/- mice had increased intestinal IL-6 expression. Intestinal lipid trafficking was altered in tis7-/- compared with WT mice postresection. In contrast, high-fat diet-fed tis7tg mice had improved survival postresection compared with WT littermates. High-fat diet feeding in the setting of tis7 deletion resulted in postresection anastomotic inflammation and small bowel obstruction. Tolerance of a calorie-rich, high-fat diet postresection may require tis7 and its target genes. The presence of luminal fat in the setting of tis7 deletion promotes an intestinal inflammatory response postresection. [ABSTRACT FROM AUTHOR]

We previously demonstrated increased villus height following genetic deletion, or knockout, of retinoblastoma protein (Rb) in the intestinal epithelium (Rb-IKO). Here we determined the functional consequences of augmented mucosal growth on intestinal fat absorption and following a 50% small bowel resection (SBR). Mice with constitutively disrupted Rb expression in the intestinal epithelium (Rb-IKO) along with their floxed (wild-type, WT) littermates were placed on a high-fat diet (HFD, 42% kcal fat) for 54 wk. Mice were weighed weekly, and fat absorption, indirect calorimetry, and MRI body composition were measured. Rb-IKO mice were also subjected to a 50% SBR, followed by HFD feeding for 33 wk. In separate experiments, we examined intestinal fat absorption in mice with conditional (tamoxifen-inducible) intestinal Rb (inducible Rb-IKO) deletion. Microarray revealed that the transcriptional expression of lipid absorption/transport genes was significantly reduced in constitutive Rb-IKO mice. These mice demonstrated greater mucosal surface area yet manifested paradoxically impaired intestinal long-chain triglyceride absorption and decreased cholesterol absorption. Despite attenuated lipid absorption, there were no differences in metabolic rate, body composition, and weight gain in Rb-IKO and WT mice at baseline and following SBR. We also confirmed fat malabsorption in inducible Rb-IKO mice. We concluded that, despite an expanded mucosal surface area, Rb-IKO mice demonstrate impaired lipid absorption without compensatory alterations in energy homeostasis or body composition. These findings underscore the importance of delineating structural/functional relationships in the gut and suggest a previously unknown role for Rb in the regulation of intestinal lipid absorption. [ABSTRACT FROM AUTHOR]

Intestinal adaptation is an important compensatory response to massive small bowel resection (SBR) and occurs because of a proliferative stimulus to crypt enterocytes by poorly understood mechanisms. Recent studies suggest the enteric nervous system (ENS) influences enterocyte proliferation. We, therefore, sought to determine whether ENS dysfunction alters resection-induced adaptation responses. Ret+/- mice with abnormal ENS function and wild-type (WT) littermates underwent sham surgery or 50% SBR. After 7 days, ileal morphology, enterocyte proliferation, apoptosis, and selected signaling proteins were characterized. Crypt depth and villus height were equivalent at baseline in WT and Ret+/- mice. In contrast after SBR, Ret+/- mice had longer villi (Ret+/- 426.7 μ± 46.0 m vs. WT 306.5 ± 7.7 μm, P < 0.001) and deeper crypts (Ret+/- 119 ± 3.4 μm vs. WT 82.4 ± 3.1 μm, P < 0.001) than WT. Crypt enterocyte proliferation was higher in Ret+/- (48.8 ± 1.3%) than WT (39.9 ± 2.1%; P < 0.001) after resection, but apoptosis rates were similar. Remnant bowel of Ret+/- mice also had higher levels of glucagon-like peptide 2 (6.2-fold, P < 0.005) and amphiregulin (4.6-fold, P < 0.001) mRNA after SBR, but serum glucagon- like peptide 2 protein levels were equal in WT and Ret+/- mice, and there was no evidence of increased c-Fos nuclear localization in submucosal neurons. Western blot confirmed higher crypt epidermal growth factor receptor (EGFR) protein levels (1.44-fold; P < 0.001) and more phosphorylated EGFR (2-fold; P < 0.003) in Ret+/- than WT mice after SBR. These data suggest that Ret heterozygosity enhances intestinal adaptation after massive SBR, likely via enhanced EGFR signaling. Reducing Ret activity or altering ENS function may provide a novel strategy to enhance adaptation attenuating morbidity in patients with short bowel syndrome. [ABSTRACT FROM AUTHOR]

Increased apoptosis in crypt enterocytes is a key feature of intestinal adaptation following massive small bowel resection (SBR). Expression of the proapoptotic factor Bax has been shown to be required for resection-induced apoptosis. It has also been demonstrated that p38-α MAPK (p38) is necessary for Bax activation and apoptosis in vitro. The present studies were designed to test the hypothesis that p38 is a key regulator of Bax activation during adaptation after SBR in vivo. Enterocyte expression of p38 was deleted by tamoxifen administration to activate villin-Cre in adult mice with a floxed Mapk14 (p38-α) gene. Proximal 50% SBR or sham operations were performed on wild-type (WT) and p38 intestinal knockout (p38-IKO) mice under isoflurane anesthesia. Mice were killed 3 or 7 days after operation, and adaptation was analyzed by measuring intestinal morphology, proliferation, and apoptosis. Bax activity was quantified by immunoprecipitation, followed by Western blotting. After SBR, p38-IKO mice had deeper crypts, longer villi, and accelerated proliferation compared with WT controls. Rates of crypt apoptosis were significantly lower in p38-IKO mice, both at baseline and after SBR. Levels of activated Bax were twofold higher in WT mice after SBR relative to sham. In contrast, activated Bax levels were reduced by 67% in mice after p38 MAPK deletion. Deleted p38 expression within the intestinal epithelium leads to enhanced adaptation and reduced levels of enterocyte apoptosis after massive intestinal resection. p38-regulated Bax activation appears to be an important mechanism underlying resection-induced apoptosis. [ABSTRACT FROM AUTHOR]

Previous studies have demonstrated that the proapoptotic protein Bax plays an important role in the elevated enterocyte apoptosis that occurs during the intestinal adaptation response to massive small bowel resection (SBR). Additionally, epidermal growth factor receptor (EGFR) activation prevents SBR-induced enterocyte apoptosis. The present study aims to delineate the relationship between EGFR activity and intestinal epithelial cell apoptosis. Treatment of model intestinal epithelial cells (RIEC-18) with both a selective EGFR inhibitor (ZD1839) and EGFR small interfering RNA knockdown resulted in a dramatic increase in apoptosis, accompanied by rapid phosphorylation of p38α. Concurrently, Bax underwent conformational changes consistent with activation and translocated to mitochondria. In contrast, EGF stimulation enhanced cell survival by attenuating p38α phosphorylation, Bax conformational change, mitochondrial trafficking, and apoptosis. These results demonstrate that that diminished EGFR activity initiates the intrinsic pathway of apoptosis through p38α-dependent Bax activation in intestinal epithelial cells. These finding provide mechanistic insight into the role that EGFR signaling plays in the regulation of enterocyte apoptosis following massive intestinal loss. [ABSTRACT FROM AUTHOR]

Previous work in our group has demonstrated that mouse salivary gland has the highest concentration of salivary-derived VEGF protein compared with other organs and is essential for normal palatal mucosal wound healing. We hypothesize that salivary VEGF plays an important role in maintaining the integrity of the gastrointestinal mucosa following small bowel resection (SBR). Thirty-five 8- to 10-wk-old C57BL/6 female mice were divided into seven treatment groups: 1) sham (transaction and anastomosis, n = 5); 2) SBR (n = 8); 3) sialoadenectomy and small bowel resection (SAL+SBR, n = 8); 4) sialoadenectomy and small bowel resection with EGF supplementation (SAL+SBR+EGF, n = 9); 5) sialoadenectomy and small bowel resection with VEGF supplementation (SAL+SBR+VEGF, n = 9); 6) sialoadenectomy and small bowel resection supplemented with EGF and VEGF (SAL+ SBR+VEGF+EGF, n = 6); 7) selective inhibition of VEGF in the submandibular gland by Ad-VEGF-Trap following small bowel resection (Ad-VEGF-Trap+SBR, n = 7). Adaptation was after 3 days by ileal villus height and crypt depth. The microvascular response was evaluated by CD31 immunostaining and for villus-vessel area ratio by FITC-labeled von Willebrand factor immunostaining. The adaptive response after SBR was significantly attenuated in the SAL group in terms of villus height (250.4 ± 8.816 vs. 310 ± 19.35, P = 0.01) and crypt depth (100.021 ± 4.025 vs. 120.541 ± 2.82, P = 0.01). This response was partially corrected by orogastric VEGF or EGF alone. The adaptive response was completely restored when both were administered together, suggesting that salivary VEGF and EGF both contribute to intestinal adaptation. VEGF increases the vascular density (6.4 ± 0.29 vs. 6.1 ± 0.29 vs. 5.96 ± 0.20) and villus-vessel area ratio (0.713 ± 0.01 vs. 0.73 ± 0.01) in the adapting bowel. Supplementation of both EGF and VEGF fully rescues adaptation, suggesting that the adaptive response may be dependent on VEGF-driven angiogenesis. These results support a previously unrecognized role for VEGF in the small bowel adaptive response. [ABSTRACT FROM AUTHOR]

The magnitude of gut adaptation is a decisive factor in determining whether patients are able to live independent of parenteral nutrition after massive small bowel loss. We previously established that the cyclin-dependent kinase inhibitor (CDKI) p21waf1/cip1 is necessary for enterocyte proliferation and a normal adaptation response. In the present study, we have further elucidated the role of this CDKI in the context of p27kip1, another member of the Cip/Kip CDKI family. Small bowel resections (SBRs) or sham operations were performed in control (C57/BL6), p21waf1/cip1-null, p27kip1-null, and p21waf1/cip1/p27kip1 double-null mice. Morphological (villus height/crypt depth) alterations in the mucosa, the kinetics of enterocyte turnover (rates of enterocyte proliferation and apoptosis), and the protein expression of various cell cycle-regulatory proteins were recorded at various postoperative times. Enterocyte compartment-specific mRNA expression was investigated using laser capture microdissection. Resection-induced adaptation in control mice coincided with increased protein expression of p21waf1/cip1 and decreased p27kip1 within 3 days postoperatively. Identical changes in mRNA expression were detected in crypt but not in villus enterocytes. Adaptation occurred normally in control and p27kip1-null mice; however, mice deficient in both p21waf1/cip1 and p27kip1 failed to increase baseline rates of enterocyte proliferation and adaptation. The expression of p21waf1/cip1 protein and mRNA in the proliferative crypt compartment is necessary for resection-induced enterocyte proliferation and adaptation. The finding that deficient expression of p27kip1 does not affect adaptation suggests that these similar CDKI family members display distinctive cellular functions during the complex process of intestinal adaptation. [ABSTRACT FROM AUTHOR]

Prior studies of intestinal adaptation after massive small bowel resection (SBR) have focused on growth factors and their effects on amplification of the gut mucosa. Because adaptive changes have also been described in intestinal smooth muscle, we sought to determine the effect of targeted smooth muscle growth factor overexpression on resectioninduced intestinal adaptation. Male transgenic mice with smooth muscle cell overexpression of insulin-like growth factor I (IGF-I) by virtue of an α-smooth muscle actin promoter were obtained. SMP8 IGF-I transgenic (IGF-I TG) and nontransgenic (NT) littermates underwent 50% proximal SBR or sham operation and were then killed after 3 or 28 days. NT mice showed the expected alterations in mucosal adaptive parameters after SBR, such as increased wet weight and villus height. The IGF-I TG mice had inherently taller villi, which did not increase significantly after SBR. In addition, IGF-I TG mice had a 50% postresection persistent increase in remnant intestinal length, which was associated with an early decline and later increase in relative mucosal surface area. These results indicate that growth factor overexpression within the muscularis layer of the bowel wall induces significant postresection adaptive intestinal lengthening and a unique mucosal response. IGF-I signaling within the muscle wall may play an important role in the pathogenesis of resection-induced adaptation. [ABSTRACT FROM AUTHOR]

Intestinal adaptation following small bowel resection (SBR) is associated with greater rates of enterocyte apoptosis by unknown mechanism(s). Because postresection adaptation is associated with increased translocation of luminal bacteria, we sought to characterize the role for the extrinsic, death receptor pathway for the activation of enterocyte apoptosis after massive SBR. We first performed SBR or sham operations in mice, and the temporal expression of caspases 8, 9, and 3, death receptors tumor necrosis factor receptor-1 (TNFR[sub 1]) and Fas and corresponding ligands (TNF and Fas ligand) was determined in the remnant intestine at various postoperative time points. Ileal TNFR[sub 1] and Fas expression were then measured after SBR in the setting of increased (waved-2 mice) or decreased (exogenous EGF administration) apoptosis. Finally, intestinal adaptation and apoptosis were recorded in the remnant ileum after SBR in TNFR[sub 1]-null and Fas-null mice. The expression of death receptor family proteins and caspases demonstrated only modest changes after SBR and did not correlate with the histological appearance of apoptosis. In the setting of accelerated apoptosis, TNFR[sub 1] and Fas expression were paradoxically decreased. Apoptotic and adaptive responses were preserved in both TNFR[sub 1]-null and Fas-null mice. These results suggest that the mechanism for increased enterocyte apoptosis following massive SBR does not appear to involve the extrinsic, death receptor-mediated pathway. [ABSTRACT FROM AUTHOR]

Examines the role of epidermal growth factor and its receptor in chemotherapy-induced intestinal injury. Weight recovery; Evidence showing that apoptosis was not decreased; Occurrence of mucositis.