Matthew C. Koopmann

Insulin-like growth factor I and glucagon-like peptide-2 responses to fasting followed by controlled or ad libitum refeeding in rats

Luminal nutrients stimulate structural and functional regeneration in the intestine through mechanisms thought to involve insulin-like growth factor I (IGF-I) and glucagon-like peptide-2 (GLP-2). We investigated the relationship between IGF-I and GLP-2 responses and mucosal growth in rats fasted for 48 h and then refed for 2 or 4 days by continuous intravenous or intragastric infusion or ad libitum feeding. Fasting induced significant decreases in body weight, plasma concentrations of IGF-I and bioactive GLP-2, jejunal mucosal cellularity (mass, protein, DNA, and villus height), IGF-I mRNA, and ileal proglucagon mRNA. Plasma IGF-I concentration was restored to fed levels with 2 days of ad libitum refeeding but not with 4 days of intravenous or intragastric refeeding. Administration of an inhibitor of endogenous GLP-2 (rat GLP-2 3-33) during ad libitum refeeding partially attenuated mucosal growth and prevented the increase in plasma IGF-I to fed levels; however, plasma GLP-2 and jejunal IGF-I mRNA were restored to fed levels. Intragastric refeeding restored intestinal cellularity and functional capacity (sucrase activity and sodium-glucose transporter-1 expression) to fed levels, whereas intravenous refeeding had no effect. Intestinal regeneration after 4 days of intragastric or 2 days of ad libitum refeeding was positively associated with increases in plasma concentrations of GLP-2 and jejunal IGF-I mRNA. These data suggest that luminal nutrients stimulate intestinal growth, in part, by increased expression of both GLP-2 and IGF-I.

Exogenous Glucagon-Like Peptide-2 (GLP-2) Augments GLP-2 Receptor mRNA and Maintains Proglucagon mRNA Levels in Resected Rats

BACKGROUND Glucagon-like peptide-2 (GLP-2) is a nutrient-dependent proglucagon-derived hormone that stimulates intestinal adaptive growth. Our aim was to determine whether exogenous GLP-2 increases resection-induced adaptation without diminishing endogenous proglucagon and GLP-2 receptor expression. METHODS Rats underwent transection or 70% jejunoileal resection +/- GLP-2 infusion (100 microg/kg body weight/d) and were fed a semipurified diet with continuous infusion of GLP-2 or saline by means of jugular catheter. After 7 days, body weight, mucosal cellularity (dry mass, protein and DNA), crypt-villus height, and crypt cell proliferation (by bromodeoxyuridine staining) were determined. Plasma bioactive GLP-2 (by radioimmunoassay), proglucagon and GLP-2 receptor mRNA expression (by Northern blot and real-time reverse transcriptase quantitative polymerase chain reaction) were measured. GLP-2 receptor was colocalized to neuroendocrine markers by immunohistochemistry. RESULTS Low-dose exogenous GLP-2 increased mucosal cellularity and crypt-villus height in the duodenum, jejunum, and ileum; enterocyte proliferation in the jejunal crypt; and duodenal and jejunal sucrase segmental activity. Plasma bioactive GLP-2 concentration increased 70% upon resection, with an additional 54% increase upon GLP-2 infusion in resected rats (P < .05). Ileal proglucagon mRNA expression increased with resection, and exogenous ileum GLP-2 failed to blunt this response. Exogenous GLP-2 increased ileum GLP-2 receptor expression 3-fold in resected animals and was colocalized to vasoactive intestinal peptide-positive and endothelial nitric oxide synthase-expressing enteric neurons and serotonin-containing enteroendocrine cells in the jejunum and ileum of resected rats. CONCLUSIONS Exogenous GLP-2 augments adaptive growth and digestive capacity of the residual small intestine in a rat model of mid-small bowel resection by increasing plasma GLP-2 concentrations and GLP-2 receptor expression without diminishing endogenous proglucagon expression.

Laparoscopic and Minimally Invasive Resection of Malignant Colorectal Disease

Minimally invasive surgery for colorectal cancer is a burgeoning field of general surgery. Randomized controlled trials have assessed short-term patient-oriented and long-term oncologic outcomes for laparoscopic resection. These trials have demonstrated that the laparoscopic approach is equivalent to open surgery with a shorter hospital stay. Laparoscopic resection also may result in improved short-term patient-oriented outcomes and equivalent oncologic resections versus the open approach. Transanal excision of select rectal cancer using endoscopic microsurgery is promising and robotic-assisted laparoscopic surgery is an emerging modality. The efficacy of minimally invasive treatment for rectal cancer compared with conventional approaches will be clarified further in randomized controlled trials.

Sustained glucagon-like peptide-2 infusion is required for intestinal adaptation, and cessation reverses increased cellularity in rats with intestinal failure

Glucagon-like peptide-2 (GLP-2) is a nutrient-dependent, proglucagon-derived hormone that is a proposed treatment for human short bowel syndrome (SBS). The objective was to determine how the timing, duration, and cessation of GLP-2 administration affect intestinal adaptation and enterocyte kinetics in a rat model of human SBS that results in intestinal failure requiring total parenteral nutrition (TPN). Rats underwent 60% jejunoileal resection plus cecectomy and jugular vein cannulation and were maintained exclusively with TPN for 18 days in these treatments: TPN control (no GLP-2); sustained GLP-2 (1-18 days); early GLP-2 (1-7 days, killed at 7 or 18 days); and delayed GLP-2 (12-18 days). Body weight gain was similar across groups, and plasma bioactive GLP-2 was significantly increased with coinfusion of GLP-2 (100 μg·kg⁻¹·day⁻¹) with TPN. GLP-2-treated rats showed significant increases in duodenum and jejunum mucosal dry mass, protein, DNA, and sucrase activity compared with TPN control. The increased jejunum cellularity reflected significantly decreased apoptosis and increased crypt mitosis and crypt fission due to GLP-2. When GLP-2 infusion stopped at 7 days, these effects were reversed at 18 days. Sustained GLP-2 infusion significantly increased duodenum length and decreased 18-day mortality to 0% from 37.5% deaths in TPN control (P = 0.08). Colon proglucagon expression quantified by real-time RT-qPCR was increased in TPN controls and attenuated by GLP-2 infusion; jejunal expression of the GLP-2 receptor did not differ among groups. In summary, early, sustained GLP-2 infusion reduces mortality, induces crypt fission, and is required for intestinal adaptation, whereas cessation of GLP-2 reverses gains in mucosal cellularity in a rat model of intestinal failure.

Colonic GLP-2 is not Sufficient to Promote Jejunal Adaptation in a PN-Dependent Rat Model of Human Short Bowel Syndrome

BACKGROUND Bowel resection may lead to short bowel syndrome (SBS), which often requires parenteral nutrition (PN) due to inadequate intestinal adaptation. The objective of this study was to determine the time course of adaptation and proglucagon system responses after bowel resection in a PN-dependent rat model of SBS. METHODS Rats underwent jugular catheter placement and a 60% jejunoileal resection + cecectomy with jejunoileal anastomosis or transection control surgery. Rats were maintained exclusively with PN and killed at 4 hours to 12 days. A nonsurgical group served as baseline. Bowel growth and digestive capacity were assessed by mucosal mass, protein, DNA, histology, and sucrase activity. Plasma insulin-like growth factor I (IGF-I) and bioactive glucagon-like peptide 2 (GLP-2) were measured by radioimmunoassay. RESULTS Jejunum cellularity changed significantly over time with resection but not transection, peaking at days 3-4 and declining by day 12. Jejunum sucrase-specific activity decreased significantly with time after resection and transection. Colon crypt depth increased over time with resection but not transection, peaking at days 7-12. Plasma bioactive GLP-2 and colon proglucagon levels peaked from days 4-7 after resection and then approached baseline. Plasma IGF-I increased with resection through day 12. Jejunum and colon GLP-2 receptor RNAs peaked by day 1 and then declined below baseline. CONCLUSIONS After bowel resection resulting in SBS in the rat, peak proglucagon, plasma GLP-2, and GLP-2 receptor levels are insufficient to promote jejunal adaptation. The colon adapts with resection, expresses proglucagon, and should be preserved when possible in massive intestinal resection.

Total parenteral nutrition attenuates cerulein-induced pancreatitis in rats.

Objectives: Our aim was to determine if total parenteral nutrition (TPN)-induced pancreatic atrophy and Hsp70 expression attenuates cerulein-induced pancreatitis in rats. Methods: Rats were randomized to a 7-day course of saline infusion plus a semipurified diet or TPN, with or without an intravenous cerulein injection or vehicle on day 7, and killed 1 or 6 hours after the injection. Based on a pilot study, 1 hour was the primary time point. Pancreatic atrophy was determined by mass, protein, and DNA contents. Pancreatic heat shock protein 70 (Hsp70) expression was measured by Western analysis. Histological examination of the pancreas assessed for edema, inflammation, vacuolization, and apoptosis. Serum amylase activity was measured using the Phadebas assay. Pancreatic trypsinogen activation was measured using a fluorometric substrate assay. Results: The saline-infused rats fed orally gained significantly more weight than TPN rats. The TPN decreased the pancreatic mass and protein content and the protein-DNA ratio and increased the pancreatic DNA content compared with the saline. The TPN increased the pancreatic Hsp70 expression by 91% compared with the saline. The TPN reduced the cerulein-induced pancreatic histological edema, the vacuolization, and the inflammation compared with the saline. The increase in the serum amylase level after cerulein injection was significantly attenuated, and trypsinogen activation was reduced in TPN animals compared with the saline group. Conclusions: Lack of luminal nutrients with a 7-day course of TPN provides moderate protection against cerulein-induced pancreatitis in rats.

S1821 Differential Effects of a Protein Free Diet and Amino Acid Infusion On Pancreatic Growth and Digestive Enzyme Expression

The desmoplastic/stromal reaction in pancreatic cancer plays a critical role in tumour growth and metastasis. The key cells responsible for this fibrous reaction in pancreatic cancer are pancreatic stellate cells (PSCs). We have shown a bi-directional interaction between PSCs and cancer cells which facilitates cancer progression. Growth factors and cytokines produced by tumour cells activate PSCs (increased proliferation, migration and synthesis of extracellular matrix [ECM] proteins). We propose a role for heat shock proteins (HSPs) in these processes. HSPs are a family of chaperone proteins known to play a role in cellular functions such as apoptosis, migration and ECM modulation. We have shown that PSCs express HSP 27, 47, 70 and 90. However, little is known about their role in PSC functions. Aim: To determine whether HSP expression within PSCs is influenced by PSC activation. Methods: PSCs were isolated from pancreatic tissue resected from patients with benign pancreatic diseases (normal PSCs) and pancreatic ductal adenocarcinoma (cancer-associated, CA-PSCs). CA-PSCs were exposed to PDGF and TGFβ for 24h. HSP levels in PSCs and human pancreatic cancer cells (MiaPaCa-2, Panc1, ASPC-1) were assessed by western blotting (densitometry units, mean±SE). Results: CA-PSCs exhibited higher HSP levels compared to normal PSCs (Table). CA-PSCs also exhibited increased HSP27 and HSP47 expression compared to pancreatic cancer cells (Table). Treatment of CA-PSCs with PDGF induced HSPs (% of control data: HSP27 389±121*, HSP47 158±9*, HSP70 220±25*, HSP90 228±29*; *p<0.05). PSCs treated with TGFβ had increased HSP47 levels (152±12*, *p<0.05). Conclusions: i) CAPSCs exhibit increased HSP levels compared to normal PSCs and higher HSP27 and HSP47 levels compared to pancreatic cancer cells; and ii) HSP expression is increased in PSCs exposed to activating factors. Modulation of HSPs in PSCs may alter their function and how they interact with cancer cells.