Mildred Yim

The Role of Liver Fructose-1,6-Bisphosphatase in Regulating Appetite and Adiposity

Liver fructose-1,6-bisphosphatase (FBPase) is a regulatory enzyme in gluconeogenesis that is elevated by obesity and dietary fat intake. Whether FBPase functions only to regulate glucose or has other metabolic consequences is not clear; therefore, the aim of this study was to determine the importance of liver FBPase in body weight regulation. To this end we performed comprehensive physiologic and biochemical assessments of energy balance in liver-specific transgenic FBPase mice and negative control littermates of both sexes. In addition, hepatic branch vagotomies and pharmacologic inhibition studies were performed to confirm the role of FBPase. Compared with negative littermates, liver-specific FBPase transgenic mice had 50% less adiposity and ate 15% less food but did not have altered energy expenditure. The reduced food consumption was associated with increased circulating leptin and cholecystokinin, elevated fatty acid oxidation, and 3-β-hydroxybutyrate ketone levels, and reduced appetite-stimulating neuropeptides, neuropeptide Y and Agouti-related peptide. Hepatic branch vagotomy and direct pharmacologic inhibition of FBPase in transgenic mice both returned food intake and body weight to the negative littermates. This is the first study to identify liver FBPase as a previously unknown regulator of appetite and adiposity and describes a novel process by which the liver participates in body weight regulation.

p-21-Activated kinase 1 mediates gastrin-stimulated proliferation in the colorectal mucosa via multiple signaling pathways

Gastrins, including amidated (Gamide) and glycine-extended (Ggly) forms, function as growth factors for the gastrointestinal mucosa. The p-21-activated kinase 1 (PAK1) plays important roles in growth factor signaling networks that control cell motility, proliferation, differentiation, and transformation. PAK1, activated by both Gamide and Ggly, mediates gastrin-stimulated proliferation and migration, and activation of β-catenin, in gastric epithelial cells. The aim of this study was to investigate the role of PAK1 in the regulation by gastrin of proliferation in the normal colorectal mucosa in vivo. Mucosal proliferation was measured in PAK1 knockout (PAK1 KO) mice by immunohistochemistry. The expression of phosphorylated and unphosphorylated forms of the signaling molecules PAK1, extracellular signal-regulated kinase (ERK), and protein kinase B (AKT), and the expression of β-catenin and its downstream targets c-Myc and cyclin D1, were measured in gastrin knockout (Gas KO) and PAK1 KO mice by Western blotting. The expression and activation of PAK1 are decreased in Gas KO mice, and these decreases are associated with reduced activation of ERK, AKT, and β-catenin. Proliferation in the colorectal mucosa of PAK1 KO mice is reduced, and the reduction is associated with reduced activation of ERK, AKT, and β-catenin. In compensation, antral gastrin mRNA and serum gastrin concentrations are increased in PAK1 KO mice. These results indicate that PAK1 mediates the stimulation of colorectal proliferation by gastrins via multiple signaling pathways involving activation of ERK, AKT, and β-catenin.

Protective effect of zinc preconditioning against renal ischemia reperfusion injury is dose dependent

Objectives Ischemia-reperfusion injury (IRI) is a major cause of acute kidney injury and chronic kidney disease. Two promising preconditioning methods for the kidney, intermittent arterial clamping (IC) and treatment with the hypoxia mimetic cobalt chloride, have never been directly compared. Furthermore, the protective efficacy of the chemically related transition metal Zn2+ against renal IRI is unclear. Although Co2+ ions have been shown to protect the kidney via hypoxia inducible factor (HIF), the effect of Zn2+ ions on the induction of HIF1α, HIF2α and HIF3α has not been investigated previously. Materials and methods The efficacy of different preconditioning techniques was assessed using a Sprague-Dawley rat model of renal IRI. Induction of HIF proteins following Zn2+ treatment of the human kidney cell lines HK-2 (immortalized normal tubular cells) and ACHN (renal cancer) was measured using Western Blot. Results Following 40 minutes of renal ischemia in rats, cobalt preconditioning offered greater protection against renal IRI than IC as evidenced by lower peak serum creatinine and urea concentrations. ZnCl2 (10 mg/kg) significantly lowered the creatinine and urea concentrations compared to saline-treated control rats following a clinically relevant 60 minutes of ischemia. Zn2+ induced expression of HIF1α and HIF2α but not HIF3α in HK-2 and ACHN cells. Conclusion ZnCl2 preconditioning protects against renal IRI in a dose-dependent manner. Further studies are warranted to determine the possible mechanisms involved, and to assess the benefit of ZnCl2 preconditioning for clinical applications.

Depletion of p21-activated kinase 1 up-regulates the immune system of APC∆14/+ mice and inhibits intestinal tumorigenesis

BackgroundP21-activated kinase 1 (PAK1) stimulates growth and metastasis of colorectal cancer (CRC) through activation of multiple signalling pathways. Up-regulation of CRC stem cell markers by PAK1 also contributes to the resistance of CRC to 5-fluorouracil. The aim of this study was to investigate the effect of PAK1 depletion and inhibition on the immune system and on intestinal tumour formation in APC∆14/+ mice.MethodsThe PAK1 KO APC∆14/+ mice were generated by cross-breeding of PAK1 KO mice with APC∆14/+ mice. Splenic lymphocytes were analysed by flow cytometry, and immunohistochemical staining. The numbers of intestinal tumours were counted. Blood cells were also counted.ResultsCompared to APC+/+ mice, the numbers of both T- and B- lymphocytes were reduced in the spleen of APC∆14/+ mice. Depletion of PAK1 in APC∆14/+ mice increased the numbers of splenic T- and B- lymphocytes and decreased the numbers of intestinal tumours. Treatment of APC∆14/+ mice with PF-3758309, a PAK inhibitor reduced the numbers of intestinal tumours and increased the numbers of blood lymphocytes.ConclusionDepletion of active PAK1 up-regulates the immune system of APC∆14/+ mice and suppresses intestinal tumour development. These observations suggest an important role for PAK1 in the immune response to tumours.

The C-terminal flanking peptide of progastrin induces gastric cell apoptosis and stimulates colonic cell division in vivo.

Progastrin (PG) is processed into a number of smaller peptides including amidated gastrin (Gamide), non-amidated glycine-extended gastrin (Ggly) and the C-terminal flanking peptide (CTFP). Several groups have reported that PG, Gamide and Ggly are biologically active in vitro and in vivo, and are involved in the development of gastrointestinal cancers. CTFP is bioactive in vitro but little is known of its effects in vivo. This study investigated the bioactivity of CTFP in vivo in normal tissues using gastrin deficient (GASKO) mice and in two mouse models of cancer (SCID mice bearing xenograft tumors expressing normal or knocked-down levels of gastrin and a mouse model of hepatic metastasis). As with Ggly, CTFP treatment stimulated colonic proliferation in GASKO mice compared to control. CTFP also significantly increased apoptosis in the gastric mucosa of male GASKO mice. CTFP did not appear to effect xenograft growth or the incidence of liver metastases. This is the first demonstration that CTFP has specific biological activity in vivo in the colon and stomach.

Demonstration and biological significance of a gastrin‐P21‐activated kinase 1 feedback loop in colorectal cancer cells

Gastrins, including amidated gastrin17 and glycine‐extended gastrin17, are important growth factors in colorectal cancer (CRC). The p21‐activated kinase 1 (PAK1) plays key roles in cellular processes including proliferation, survival, and motility, and in cell transformation and tumor progression. PAK1 expression increases with the progression of CRC, and knockdown of PAK1 blocks CRC cell growth and metastasis both in vitro and in vivo. The aim of this study was to determine the interaction between PAK1 and gastrins in CRC cells. PAK1 expression and activation were assayed by Western blots, and concentrations of gastrin mRNA and peptides by real‐time PCR and radioimmunoassay, respectively. Proliferation of CRC cells was measured by 3H‐thymidine incorporation, and vascular endothelial growth factor (VEGF) secretion was measured by ELISA. Gastrins activated PAK1 via PI3K‐dependent pathways. Activated PAK1 in turn mediated gastrin‐stimulated activation of β‐catenin and VEGF secretion in CRC cells, as knockdown of PAK1 blocked stimulation of these cellular processes by gastrins. Downregulation of gastrin reduced the expression and activity of PAK1, but in contrast there was a compensatory increase in gastrins either when PAK1 was downregulated, or after treatment with a PAK inhibitor. Our results indicate that PAK1 is required for the stimulation of CRC cells by gastrins, and suggest the existence of an inhibitory feedback loop by which PAK1 downregulates gastrin production in CRC cells.

S1695 In Vivo Analysis of Mouse Gastrin Gene Expression by Bac Transgenic EGFP Reporter Mice

overexpressed Plexin B1 compared to normal pancreas tissues. Endogenous Plexin B1 and Met protein were detected in eight pancreatic cancer cell lines, with a variety of expression levels. Sema 4D stimulation activated Met protein, and significantly induced cell migration. Discussion: Here we show that Sema 4D involves in the regulation of cell motility through its receptor Plexin B1. And the results of immunostaining suggest that Sema 4D could play a role in the interaction between tumor cells and tumor microenvironment in pancreatic cancer tissues. As many attempts are now ongoing to target the tumor stroma, these findings provide a new insight into molecular targeted therapies for pancreatic cancer.

Abstract 4159: Progastrin inhibits Helicobacter-associated gastric corpus carcinogenesis in mice

Background & Aims: We have previously reported that overexpression of amidated gastrin in transgenic (INS-GAS) mice with Helicobacter felis (H. felis) infection accelerated gastric corpus carcinogenesis, while overexpression of non-amidated glycine-extended gastrin in transgenic (MTI-G-gly) mice inhibited parietal cell loss and atrophy in stomach. We have also reported that overexpression of the full length progastrin in transgenic (hGAS) mice stimulated colon carcinogenesis in azoxymethane-treated mice. In this study, we investigated the role of progastrin for Helicobacter-associated gastric carcinogenesis in mice. Methods: hGAS, INS-GAS and non-transgenic wild type (B6 wt) mice on a C57BL/6 background were infected with H. felis for 12 or 18 months (m). We also analyzed male hGAS, INS-GAS and wild type (FVB wt) mice on a FVB/N background with H. felis infection for 7 m. Infection status was assessed by realtime PCR, microscopic evaluation and serum titer of H. felis-specific IgG antibodies. Proliferating cells and parietal cells were investigated by immunohistochemistry (IHC) with Ki-67 and HK-ATP-beta antibodies, respectively. Expression of stem cell-related genes such as CD44 & DCAMKL-1 was also analyzed by IHC. Results: At 12 m post infection (p.i.), INS-GAS mice had mild corpus dysplasia and B6 wt mice had severe gastritis or metaplasia, while hGAS mice had only mild to moderate gastritis, respectively. At 18 m p.i., both INS-GAS and B6 wt mice had severe atrophic gastritis and corpus dysplasia, while hGAS mice showed only moderate gastritis with mild gastric atrophy but no corpus dysplasia. In contrast, hGAS mice had moderate to severe antral or pyloric dysplasia, while INS-GAS and B6 wt mice did not. H. felis colonization remained stable over time among 3 groups of mice. The serum titer of H. felis-specific IgG antibody as well as Th1-Th2 polarization calculated by IgG1/IgG2c subclass ratio showed no significant differences among 3 groups. At 18 m p.i., the number of Ki-67(+) cells per gastric corpus gland was the highest in INS-GAS and the lowest in hGAS mice. Parietal cell loss was remarkable in INS-GAS and B6 wt, but minimal in hGAS mice. CD44(+) and DCAMKL-1(+) cell numbers were significantly increased in INS-GAS and B6 wt, while essentially unchanged in hGAS mice. On a FVB/N background, INS-GAS mice with H. felis infection for 7 m showed gastric corpus adenocarcinoma, and infected FVB wt mice showed severe gastric atrophy with intestinal metaplasia, while infected hGAS mice showed only moderate gastritis without metaplasia or dysplasia. Conclusions: These results point to a protective effect of progastrin on Helicobacter-associated gastric corpus carcinogenesis perhaps by inhibiting the loss of parietal cells. In contrast antral pathology is increased in hGAS mice. We conclude that the different forms of gastrin have contrasting effects on Helicobacter-associated gastric corpus carcinogenesis in mice. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 4159.