Advanced glycation end products accumulate in the diabetic gallbladder

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ASSOCIATION FOR ACADEMIC SURGERY AND SOCIETY OF UNIVERSITY SURGEONS—ABSTRACTS DIFFERENTIATION IN NORMAL LIVER AND MAY PROTECT FROM INSULIN INDUCED STEATOSIS IN THE LIVER. K. Kitisin, W. Jogunoori, Y. Tang, V. Katuri, K. Shetty, B. Mishra, L. Mishra, L. Johnson; Georgetown University, Washington, DC.

Over the last decade, the prevalence of obesity in Western countries has more than doubled. Fatty liver is correlated with increases in obesity and type II diabetes. Steatosis was regarded as benign in the past, but now there is growing evidence that steatosis represents a progression of liver fibrosis and liver injury. Clinically, steatosis can be associated with wide range of hepatic dysfunction ranging from asymptomatic elevated liver enzymes to cirrhosis with complications of liver failure and possibly, the development of hepatocellular carcinoma (HCC). Disruption of the transforming growth factorBeta (TGF-Beta) signaling cascade, which plays a critical role in hepatic homeostasis and normal liver development, might contribute to initiation of hepatic steatosis as well. Aside from the TGF-Beta signaling pathway, other proteins, including forkhead (FOXO) transcriptional factors, are needed to ensure normal hepatic and biliary epithelial cell development. Precise modulation of TGF-Beta through Smad activation by the type I receptors is dependent upon adaptor proteins such as ELF, a beta-spectrin. Normal biliary epithelial development is not seen in the elf -/- mutant embryos, which have a similar phenotype to Smad2⫹/⫺/3⫹/⫺ mutants. In addition, Smad2⫹/⫺/3⫹/⫺ mice die at mid-gestation from defective liver development. Aims: We utilized a model system, embryonic liver explant cultures of the embryonic mouse (E 11.5), to determine the role of TGF-Beta signaling in liver progenitor cell proliferation and differentiation. Methods and Results: Embryonic liver tissues isolated from normal (wt), elf⫹/⫺ and Smad2⫹/⫺/3⫹/⫺ mutants were isolated and cultured in a large range of growth factor combinations and conditions to determine their growth properties. 1. Culture explants from Smad2⫹/⫺/3⫹/⫺ mutants showed a lack of bile ductules, and absent labeling for cytokeratins. 2. Addition of TGFBeta produced a rise in Smad2 and 3 activity, expression occurring at the rapid outgrowth of a limiting plate with bile duct formation, that labeled positively for ck14 and ck19, in Smad2⫹/⫺/3⫹/⫺ mutant embryos. 3. Explants from Smad2⫹/⫺/3⫹/⫺ and elf⫹/⫺ mutant embryos treated with insulin demonstrated a dramatic increase in steatosis. Conclusions: TGF-Beta signaling is required for normal intrahepatic bile duct formation and in the recruitment of the hepatic epithelium and mesenchyme at early time-points of liver and bile duct development. Additionally, the disruption of TGF-Beta signaling by alteration of Smad2/3 or ELF proteins inhibits normal differentiation of bipotential cells and results in abnormal bile duct formation. In Smad2⫹/⫺/3⫹/⫺ and elf⫹/⫺ mutant embryo explants, the addition of insulin resulted in severe steatosis. The mechanisms of these findings could be secondary to the alteration in FOXO factors level, whereby FOXO factors are sequestered by Insulin driven Akt, and are unable to partner with Smads to exert TGF-Beta cytostatic and tumor suppressor functions. More importantly, in adult tissues these derangements may promote fibrosis and hepatocarcinogenesis. 389. ADVANCED GLYCATION END PRODUCTS ACCUMULATE IN THE DIABETIC GALLBLADDER. D. A. Swartz-Basile 1, D. Lu 1, K. Q. Tran 2, S. Graewin 2, A. Nakeeb 1, H. A. Pitt 1; 1Indiana University School of Medicine, Indianapolis, IN, 2Medical College of Wisconsin, Milwaukee, WI. Introduction: Diabetes is a common condition which is associated with multiple comorbidities including gallstones. We recently demonstrated that diabetic mouse strains including, leptin-deficient (Lep ob), leptin-resistent (Lep db) and non-obese diabetic (NOD) mice, have impaired biliary motility and that administration of leptin to Lep ob mice and ciliary neurotrophic factor to Lep db mice ameliorates this response. We have also demonstrated that gallbladder

myocytes from Lep ob diabetic, obese mice are foreshortened and respond poorly to cholecystokinin. Advanced glycation end products (AGEs), the derivatives of glucose-protein and glucose-lipid interactions, have been implicated in diabetes-related complications. Recent studies have demonstrated the accumulation of AGE-modified proteins in several human tissues including renal proximal tubules in patients with diabetic nephropathy, retina, peripheral nerves, atherosclerotic lesions and senile plaques in patients with Alzheimer’s disease. Therefore, we hypothesized that diabetes-associated accumulation of AGEs in the gallbladder may explain alterations in gallbladder function. Therefore, the aim of this study was to compare the level of AGE in the gallbladder and liver of non-diabetic, lean and diabetic, obese mice. Methods: Eight week old, nondiabetic C57BL/6J (n⫽7) and diabetic Lep db (n⫽7) female mice were fed a chow diet for four weeks. At 12 weeks of age, all mice were fasted overnight and underwent cholecystectomy and hepatectomy. Presence of AGE was determined in gallbladder and liver by competitive ELISA using a monoclonal antibody raised against N-(carboxymethyl)lysine (CML), an established biomarker for AGEs. Resulting data were analyzed using Student’s t-test. Results: AGE was significantly higher in the gallbladder of diabetic Lep db mice compared to nondiabetic C57BL/6J control mice. However, AGE was significantly lower in the liver of diabetic mice compared to controls. Results are shown in the table below. Conclusions: These data suggest that advanced glycation end products accumulate in the diabetic gallbladder and that the diabetic liver may have a protective mechanism to prevent AGE accumulation. Therefore, we conclude that the presence of advanced glycation end products in the gallbladder wall may play a role in diabetic biliary dyskinesia and gallstone formation. Gallbladder (␮g/g protein)

Liver (␮g/g protein)

C57BI/6J (n ⫽ 7) Lep db (n ⫽ 6) C57BI/6J (n ⫽ 6) Lep db (n ⫽ 7) 12.7 ⫾ 1.8 81.7 ⫾ 10.4ⴱ 10.3 ⫾ 2.5 3.6 ⫾ 1.1†‡ Data are means ⫾ SEM. ⴱp⬍0.0001 vs C57BI/6J gallbladder, †p⬍0.05 vs C57BI/6J liver, ‡p⬍0.000001 vs Lep db gallbladder 390. OPEN SURGICAL CLIPPING OF MURINE BILE DUCT IS MORE EFFICIENT THAN A LAPAROSCOPIC APPROACH. Kirkland J, Garrett R, Dada S, Corvera CU; Universtity of California San Francisco The purpose of this study was to generate a mouse model of biliary inflammation that minimizes post-operative death and maintains an effective inflammatory response. METHODS: Two surgical procedures were performed: an open and a laparoscopic approach. In both, the bile duct was obstructed using a mini-surgical clip placed on the distal common bile duct. Gallbladder inflammation was assessed on postoperative day 5 by hisotlogic grading and myeloperoxidase assay. RESULTS: The open technique was performed in 30 animals, 25 (83%) survived until postoperative day 5. The laparoscopic approach was performed in 5 animals, however, none survived. The open surgical approach was much more efficient than the laparoscopic approach in terms of average surgical time (14e3 min versus 72 e 11 min). Histologic evaluation of mice from the open group showed increased cellular infiltration and wall thickening. The inflamed gallbladder wall thickness increased 5 fold compared to control animals. Inflammation increased by 100 fold in the open clipped group as assessed by myeloperoxidase assay compared to controls. CONCLUSION: The open surgical model of bile duct obstruction is superior to the laparoscopic approach in the mouse. Clipping the distal bile duct is fast, simple and minimizes dissection and causes reproducible gallbladder inflammation. 391. SSTR5 REGULATES PANCREAS GENE EXPRESSION AND ISLET CELL PROLIFERATION: A MICROARRAY