David W. Lim

Glucagon-Like Peptide-2 Alters Bile Acid Metabolism in Parenteral Nutrition–Associated Liver Disease

BACKGROUND We aim to study the mechanisms underlying our previous finding that exogenous glucagon-like peptide-2 (GLP-2) treatment in a preclinical model of neonatal parenteral nutrition-associated liver disease (PNALD) improves cholestasis. METHODS Neonatal piglets received 17 days of parenteral nutrition (PN) therapy and either saline control (PN/Saline n = 8) or GLP-2 treatment at 11 nmol/kg/d (PN/GLP-2, n = 7). At terminal laparotomy, bile and liver samples were collected. The relative gene expression of enzymes involved in bile acid synthesis, regulation, and transport was measured in liver by reverse-transcriptase quantitative polymerase chain reaction. Bile acid composition in bile was determined using tandem mass spectrometry. Data were analyzed using 1-way analysis of variance (ANOVA) or Kruskal-Wallis ANOVA. RESULTS GLP-2 increased the expression of bile acid export genes: multidrug resistance-associated proteins 2 (MRP2) (P = .002) and 3 (MRP3) (P = .037) over saline control. GLP-2 increased expression of Farnesoid X receptor (FXR) (P < .001) and CYP7A1 (cytochrome P450, family 7, subfamily A, polypeptide 1) (P = .03). GLP-2 treatment was associated with decreased concentrations of taurohyocholic acid and conjugates of toxic lithocholic acid (P < .01). GLP-2 treatment increased the liver bile acid content. CONCLUSIONS GLP-2 treatment was associated with alterations in the hepatic expression of genes involved in bile acid metabolism. The transcriptomic results indicate the mechanisms at the transcriptional level acting to regulate bile acid synthesis and increase bile acid export. Differences in bile acid profiles further support a beneficial role for GLP-2 therapy in PNALD.

Emerging Piglet Models of Neonatal Short Bowel Syndrome

Short bowel syndrome (SBS) is a growing problem in the human neonatal population. In infants, SBS is the leading cause of intestinal failure, the state of being unable to absorb sufficient nutrients for growth and development. Neonates with SBS are dependent on long-term parenteral nutrition therapy, but many succumb to the complications of sepsis and liver disease. Research in neonatal SBS is challenged by the ethical limits of studying sick human neonates and the heterogeneous nature of the disease process. Outcomes in SBS vary depending on residual intestinal anatomy, intestinal length, patient age, and exposure to nutrition therapies. The neonatal piglet serves as an appropriate translational model of the human neonate because of similarities in gastrointestinal ontogeny, physiological maturity, and adaptive processes. Re-creating the disease process in a piglet model presents a unique opportunity for researchers to discover novel insights and therapies in SBS. Emerging piglet models of neonatal SBS now represent the entire spectrum of disease seen in human infants. This review aims to contextualize these emerging piglet models within the context of SBS as a heterogeneous disease. We first explore the factors that account for SBS heterogeneity and then explore the suitability of the neonatal piglet as an appropriate translational animal model. We then examine differences between the emerging piglet models of neonatal SBS and how these differences affect their translational potential to human neonates with SBS.

In a Neonatal Piglet Model of Intestinal Failure, Administration of Antibiotics and Lack of Enteral Nutrition Have a Greater Impact on Intestinal Microflora Than Surgical Resection Alone.

Background: Data are limited on how short bowel syndrome (SBS) affects the healthy developing intestinal microbiome, with even less assessing different SBS anatomical surgical models. This study was conducted to describe the “ileal” and “colonic” microflora in 2 surgical models of SBS. Materials and Methods: Neonatal piglets (2–5 days old) underwent intestinal resection, leaving the ileum (JI anatomy, n = 6) or removing the ileum and ileocecal valve (JC anatomy, n = 5), or sham surgery (sham; n = 4). JI, JC, and sham piglets commenced parenteral nutrition on day 0 and received ampicillin and trimethoprim-sulfadoxine on days 0–4 for prevention of line sepsis. At day 7, ileal and colonic digesta were collected, and they were also collected from age-matched sow-fed piglets (n = 6). DNA extraction, sequencing, and annotation followed standard procedures. Results: Colonic and ileal bacterial genus diversity and relative bacterial abundance were greater (P < .05) in sow-fed compared with JI, JC, and sham piglets; however, minor differences were observed in either location between sham, JI, and JC piglets and within the surgical model. In the colon, sow-fed piglets had higher (P < .05) abundance of Lactobacillus (26%) and tended to have lower (P = .06) abundance of Enterococcus (<.1%) than JI, JC, or sham piglets, in which Lactobacillus and Enterococcus abundance averaged <.1% and 9%, respectively. Conclusions: Intestinal resection reduces bacterial diversity in the large bowel, and the difference is associated with the presence/absence of the ileum and ileocecal valve. The lack of enteral nutrition and antibiotic administration (ie, sow-fed vs surgery) had a greater influence on the observed shift in diversity and relative abundance than intestinal resection.

Glucagon-Like Peptide 2 Improves Cholestasis in Parenteral Nutrition–Associated Liver Disease

BACKGROUND Parenteral nutrition-associated liver disease (PNALD) remains a significant cause of morbidity and mortality in neonates with intestinal failure. Although glucagon-like peptide-2 (GLP-2) is being advanced as therapy, the effect of GLP-2 treatment on PNALD is unknown. We aim to investigate the effect of exogenous GLP-2 administration on hepatic function in a neonatal piglet model of PNALD. METHODS Neonatal piglets (aged 2-6 days) underwent jugular venous catheterization to receive isonitrogenous, isocaloric parenteral nutrition (PN). Piglets were allocated to 2 groups: group 1 (n = 8) received saline while group 2 (n = 7) received GLP-2 (at 11 nmol/kg/d). After 17 days, piglets underwent terminal laparotomy, and bile flow was measured. Liver specimens were analyzed histologically and with immunoperoxidase staining. Age-matched sow-reared control piglets (group 3, n = 8) were used for comparison. RESULTS Both groups 1 and 2 receiving PN developed cholestasis relative to sow-reared controls, as evidenced by a decrease in bile flow and increase in serum total bilirubin. However, group 2 had improved bile flow (1.35 vs 0.73 µL/g; P = .02) and diminished bilirubin (38.0 vs 78.5 µmol/L; P = .008) compared with group 1. Group 2 also had lower serum alanine aminotransferase levels, a marker of liver injury. Histologically, the liver specimens in group 1 had marked hepatocyte pigmentation, which was decreased in group 2 specimens. CONCLUSIONS The exogenous administration of GLP-2 is associated with the improvement of cholestasis and liver injury. This study introduces a novel role for GLP-2 in improving PNALD in the setting of prolonged PN duration.

On the horizon: trophic peptide growth factors as therapy for neonatal short bowel syndrome.

ABSTRACT Introduction: Short bowel syndrome (SBS) occurs more commonly in human neonates than in adults. There are currently no approved therapeutic agents aimed directly at stimulating intestinal adaptation in this population. Areas Covered: A brief review of SBS and intestinal adaptation is first presented. We then present candidate peptide growth factors that are suggested to augment intestinal adaptation in SBS, with a particular focus on glucagon-like peptide-2, as well as insulin-like growth factor-1 and epidermal growth factor. The normal physiology of these peptides and our understanding of their roles in intestinal adaptation are discussed. We further consider the roles of these peptides in the ontogeny of the gastrointestinal tract and we present the limited preclinical data on the effects of administering these peptides in neonatal SBS. Expert Opinion: The clinical translation of trophic peptide therapies in neonatal SBS will require several challenges to be overcome. The optimal dose, timing and route of administration for the likely peptide, or combination of peptides, to be administered will be paramount. Despite their cost to patient care, trophic peptides have shown promise in preclinical models of neonatal SBS and may be especially beneficial for neonates that lack remnant ileum and suffer from irreversible intestinal failure.

Supplemental Parenteral Vitamin E Into Conventional Soybean Lipid Emulsion Does Not Prevent Parenteral Nutrition–Associated Liver Disease in Full-Term Neonatal Piglets

Background: Parenteral nutrition–associated liver disease (PNALD) continues to cause morbidity and mortality for neonates with intestinal failure. Lipid peroxidation is one potential etiological factor. This study was designed to test if supplementing vitamin E into conventional soy-based lipid would reduce the risk of PNALD. Methods: Sixteen piglets, aged 2–5 days and weighing 1.8–2.5 kg, were randomized to parenteral nutrition (PN) with soy lipid (SO, n = 8) or the same lipid plus &agr;-tocopherol, the most bioactive form of vitamin E (SO+E, n = 8). After 17 days, bile flow, liver chemistry, gene expression associated with bile acid metabolism, and bile acid composition were assessed. C-reactive protein (CRP) and oxidative stress markers, including plasma 8-isoprostane, were measured. All results were compared with a sow-reared control group (CON). Results: Comparing PN-treated groups, SO vs SO+E mean bile flow (5.91 vs 5.54 µL/g liver; P = .83), serum bile acid concentration (39.2 vs 26.6 µmol/L; P = .12), and total bilirubin (35.2 vs 26.9 µmol/L; P = .56) were not different. Gene expression related to bile acid metabolism and bile composition was not different between PN groups. There was no difference in CRP (41.8 vs 36.8 µg/mL; P = .22) or in plasma 8-isoprostane (27.9 vs 26.1 pg/mL; P = .77). Conclusions: In term neonatal piglets, supplemental vitamin E did not prevent cholestasis. Additional vitamin E was not associated with reduced inflammation or oxidative stress. The benefit of supplementing vitamin E into conventional lipid, vs adding fish oil, to prevent early onset of PNALD requires further clarification.

Synergy of glucagon-like peptide-2 and epidermal growth factor co-administration on intestinal adaptation in neonatal piglets with short bowel syndrome.

Glucagon-like peptide-2 (GLP-2) and epidermal growth factor (EGF) treatment enhance intestinal adaptation. To determine whether these growth factors exert synergistic effects on intestinal growth and function, GLP-2 and EGF-containing media (EGF-cm) were administered, alone and in combination, in neonatal piglet models of short bowel syndrome (SBS). Neonatal Landrace-Large White piglets were block randomized to 75% midintestinal [jejunoileal (JI) group] or distal intestinal [jejunocolic (JC) group] resection or sham control, with 7-day infusion of saline (control), intravenous human GLP-2 (11 nmol·kg-1·day-1) alone, enteral EGF-cm (80 μg·kg-1·day-1) alone, or GLP-2 and EGF-cm in combination. Adaptation was assessed by intestinal length, histopathology, Üssing chamber analysis, and real-time quantitative PCR of intestinal growth factors. Combined EGF-cm and GLP-2 treatment increased intestinal length in all three surgical models (P < 0.01). EGF-cm alone selectively increased bowel weight per length and jejunal villus height in the JI group only. The JC group demonstrated increased intestinal weight and villus height (P < 0.01) when given either GLP-2 alone or in combination with EGF-cm, with no effect of EGF-cm alone. Jejunal permeability of mannitol and polyethylene glycol decreased with combination therapy in both SBS groups (P < 0.05). No difference was observed in fat absorption or body weight gain. IGF-1 mRNA was differentially expressed in JI vs. JC piglets with treatment. Combined treatment with GLP-2 and EGF-cm induced intestinal lengthening and decreased permeability, in addition to the trophic effects of GLP-2 alone. Our findings demonstrate the benefits of novel combination GLP-2 and EGF treatment for neonatal SBS, especially in the JC model representing most human infants with SBS.NEW & NOTEWORTHY Glucagon-like peptide-2 (GLP-2) and epidermal growth factor (EGF) are intestinotrophic, with demonstrated benefit in both animal models and human studies of short bowel syndrome (SBS). The current research shows that over and above known trophic effects, the combination of GLP-2 and EGF synergistically lengthens the bowel in neonatal piglet models of SBS. Most notable benefit occurred with resection of the terminal ileum, the common clinical anatomy seen in neonatal SBS and associated with least de novo lengthening postsurgery.

Differential Effects on Intestinal Adaptation Following Exogenous Glucagon-Like Peptide 2 Therapy With and Without Enteral Nutrition in Neonatal Short Bowel Syndrome

Background: We aim to study the efficacy of exogenously administered glucagon-like peptide 2 (GLP-2) on intestinal adaptation in 2 preclinical models of neonatal short bowel syndrome (SBS) according to remnant intestinal anatomy, with and without ileum. Furthermore, we aim to determine if this adaptive effect was potentiated with enteral nutrition (EN). Methods: Neonatal piglets were block-randomized to 75% mid-intestinal (JI group, retains ileum) or distal-intestinal (JC group, has no ileum) resection or no resection (sham control) and GLP-2 treatment (11 nmol/kg/d) or saline control for 7 days. Piglets received nutrition support, either 100% parenteral nutrition (PN; 0% EN, n = 32 in total) or 80% PN + 40% EN (n = 28 in total). Adaptation was assessed by morphological and histological changes, as well as RT quantitative polymerase chain reaction of nutrient transporters and tight junctional proteins and fat absorption. Data are analyzed by 3-way analysis of variance (ANOVA) and 2-way ANOVA per EN level. Results: GLP-2 treatment lengthened villi, deepened crypts, and improved intestinal weight in the remnant intestine of JC piglets. EN was a more potent adaptive stimulus for JI piglets. Small intestinal lengthening occurred only in the JI group, when given EN. There was no difference in total fat absorption and messenger RNA expression of nutrient transporters and tight junctional proteins. Conclusions: GLP-2 administration augmented structural adaptation in JC piglets with distal intestinal resection. Given JI anatomy, further stimulation by GLP-2 treatment over innate adaptation and stimulation by EN was modest and restricted to ileum. The differential effect of GLP-2 in neonatal SBS, depending on remnant anatomy, has important implications for clinical translation and planning of clinical trials.

Impact of Clinical Use of Parenteral Lipid Emulsions on Bile Acid Metabolism and Composition in Neonatal Piglets

BACKGROUND Neonates with intestinal failure dependent on parenteral nutrition (PN) are at risk of intestinal failure-associated liver disease (IFALD). PN lipid composition relates to the risk of IFALD, but the mechanisms are poorly understood. We investigated the effects of soybean oil (SO), a mixed-lipid (ML) emulsion containing fish oil (FO), and a pure FO. We hypothesized FO-containing PN lipids would result in increased gene expression of canalicular bile acid transporters and a larger, more hydrophilic bile acid pool, predictive of increased bile flow. METHODS Neonatal piglets were allocated to receive 1 of SO, ML, or FO throughout 14 days of PN feeding. Relative expression of genes involved in bile acid synthesis and transport were determined through quantitative polymerase chain reaction. Bile secreted from the liver was collected and measured. Bile acid composition was determined using tandem mass spectrometry. Regression analysis was used to determine predictors of bile flow. RESULTS PN reduced bile acid secretion (P < .001). FO-containing PN lipids were associated with greater expression of bile acid and organic solute transport genes (P < .05) and greater secretion of hydrophobic bile acids (P < .001). Farnesoid X receptor (P = .01), bile salt export pump (P < .01), multidrug resistant protein 2 (P < .01), and unconjugated hyocholic acid (P < .001) independently predicted bile flow. CONCLUSIONS PN lipid modulation altered bile acid metabolism and composition. These alterations may explain the hepatoprotective effects of FO-containing PN lipids and support their use in the prevention and treatment of IFALD.