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Secretin (SEC) regulates expression of aquaporins (AQP)In isolated pancreatic duct cells (PDCs)
M2204
Sigmficant differences were not tound m the biophysical properties of PP responsive vs nonresponsive neurons. These data suggest that pancreatic vagal circuits controlling exocrine secretion undergo both short-term (caemlein treatment) and long-term (copper deficient diet) remodeling. Supported by NIH DK55530 and NSF 9816662.
Reduced islet regeneration alter aUoxan treatment in gastrin-CCK and gastrinTGFalpha deficient mice kennart Friis-Hansen, Troels Bock Aim: Duct to islet cell differentiation is thought to be regulated by growth factors. During pancreatic regeneration Mlowing duct ligation m rats the regenerating epithelium expresses gastrin and transforming growth factor-alpha (TGFalpha) mRNA and peptides. To examine the physiological importance of events we examined islet regeneration following alloxan treatment in wild type, gastriu deficient, gastrin-cek deficient, and gastrin TGFalpha deficient mice. Method: 20 12 weeks old mice fi'om each of the four groups of mice were give alloxan 250 mg/kg intra peritoneally. Only mice that 2 days after alloxan treatment had blood glucose above 14 mmoH were included in the study. Measurement of blood glucose and oral glucose tolerance test (OGTT) were performed once weekly during the following 8 weeks and compared to non treated controls. Resuhs: There were no difterences in body weight, weight of total pancreas or beta cell mass between the four groups of mice. Following alloxan treatment all mice were equally diabetic evaluated by fasting blood glucose and OGTT but whereas the wild type normalized their OGTT over the following 8 weeks the knockout mice had impaired normalization of OGTT. This was also reflected reduced beta cef mass (see table). Conclusion: Lack of gastrin, gastrin-CCK or gastrin-TGFalpha does not affect the development of the pancreas or the islets. However, following afloxan treatment, gastrin deficient mice had reduced islet regeneratwe ability. This was further inhibited by the additional loss of either CCK or TGFalpha. This suggests that these hormones are important for regeneration of the islets. Future expemnents will have to determine if exogenous administration of the hormones can enhance islet regeneration.
M2202
Stimulation of Pancreatic Secretion by Non-Sulfated CCK-58 Gives Insight into Neurohormonal Regulation of the Pancreas Joseph R. Reeve Jr., Mitsuyoshi Yamamoto, Gary, M. Green Non-sulfated cbolecystcMmn-58 (CCK-58ns) has been shown to be present m porcine and canine intestine, This form ot cholecystokinin may have been ignored by our laboratory in previous studies because it reacts very poorly in our radioimmunoassay with antibody 5135 (about 10% as potent as CCK-8s. However, it is present in the intestine at about half the concentration of CCK-58s. A maior bi-product from the synthesis and purification of rat CCK-58s is CCK-58ns. Syrabenc CCK-58ns was wall separated from CCK-58s, and was used to stinmlate pancreatic secretion in awake rats. CCK-58ns was as potent and one-half as effacions as CCK.-58s for stimulation ot pancreatic find and protein. CCK-8s was as potent as the CCK-58 peptides for stimulation of prutem, but CCK-8 did not stimulate fluid secretion (Figure). A model that would account tbr these results is one where both the acmar CCK-A receptor and the CCK-A receptor on intrapancreatic nerves regulate pancreatic secretion. The nanral CCK-A receptor is mediated by a neumtransmttter that can be blocked by atropine and it stinmlates both protein and fuid. We propose that this neural receptor is not activated by CCK-8s at any concetrations, possibly because CCK-8s is inactivated by neural endopeptidease before it reaches the neural CCK-A receptor. This is supported by the tact that CCK-58s is resistant to this endopepfidase degradation while CCK-8s is rapidly cleaved. We barther propose that the CCK-A receptor m~ aciuar cells is not activated by CCK-58ns and its inability to react with this receptor causes the decreased efficacy compared to CCK-58s. In conclusion, CCK-58ns is present in intestine and it has a unique pattern for stimulating pancreanc secretion that leads to new hypotheses about regulation of pancreatic secretion. -vllp- CC g 69 btS)
.-~
Wildtype
Gastdn-TGFalpha KO
Weight of pancreas (milltgrams) (day 0) Be~ cell ma~ (micrograms) {day 0) Futlng blood glucose
204~19
208~10
192:10
22125
773105
795143
752133
76789
7,70,5
8,5,10
10.8:0,9
123,0,7
W ~ t of pan~'tn~ (milli-
243,30
23414
26715
227~t4
cell mass (micrograms) (8 weeks)
690 78
584,82
370~61
442~60
(rmol/I) (8 weeks)
CO Ka (s)
Gastrln KO Gu~n.CCK
M2205
Pancreatic Reg Is a Modulator of Acinar Cell Differentiation: Influence on Cellular Lineage Didier Sanchez, Catherine Muller, Michael Zeniboan
Minutes
BACKGROUND & AIMS: Type I Reg gent, an acinar cell product, has been implicated in islet growth and maiutainence. Since the role of this acinar product in formation of islets is unclear, we tested if levels of reg l gent expression would influence acinar cellular differentiation. METHODS: We generated clones of AR42j cells, an amphicrine rat acinar pancreatic tumor line which expresses reg 1. We transfected it with a plasmid containing the entire coding sequence driven by a CMV promoter, and isolated stable clones with cDNA in sense (SS) or antisense (AS) orientation. Western blotting and Real Time-PCR were applied. RESULTS: Increased or reduced expression of Reg I mRNA and protein were confirmed. Morphologic analysis cells demonstrated more acinar-like differentiation in SS and a less differentiated state in AS. Amylase mRNA and protein levels increased in SS cells, whereas not protein levels changed in AS excepted mRNA levels decreased. AS cells showed increased cytokeratin proteins (CK-7 and 19), PDX-1 and insulin mRNAs. Conversely cytokeratin and PDX-1 decreased in SS cells (Table) CONCLUSIONS: These data demonstrated that reg I over-expression is linked to aciuar cell differentiation, and inhibition of reg l leads to the same acinar cells expressing ductal and [betal-cdl properties. We postulate that reg expression in acinar cells is important in maintaining pancreatic cell lineage, and as it changes, cells can dedifferentiate into ductal and/or [betal-cells. Relative expression of mRNAs.
M2203
Secretin (SEC) Regulates Expression of Aquaporins (AQP)In Isolated Pancreatic Duct Cells (PDCs) Carlos Mvarez, Christopher Waskewich The human pancreas, specifically duct cells, makes 1L of fluid daily in response to SEC. This process is not well defined at the cellular level Fluid secretion across epithelia is generally considered a passive, paracdlular phenomenon generated by osmotic gradients, but the recent discovery of AQPs, a differentially" expressed family of water channels, suggests that fluid secretion may be a well-regulated process in tissues like the b~ncreas with high secretory rates. We sought to define the pattern of AQP expression in PDCs and hypothesized that this would be under SEC control. METHODS: Segments of bovine main PD were harvested flesh, and maintained in explant cuhure lot 72h in basal media, The explants were tben exposed to SEC (0, 10 or 100nM). Epithelial cells were scraped off the PD connective tissue, lysed, and their protein and mRNA extracted. Western blot analysis was performed using antibodies against AQPI, 2, 4 and 8. RT-PCR was performed using primers for AQP1, with GAPDH used as housekeeper. RESULTS: AQP 1,4 and 8 protein was present in cultured duct cells, while AQP2 was not observed in the presence or absence of SEC. SEC stimulation did not aff?ct AQP protein levels at 24h after stimulation. AQP 1 RNA levels were initially depressed with exposure to SEC but recovered to levels above baseline (see char0. CONCLUSIONS: The pancreatic duct epithelium expresses an assortmem of AQPs that are likely invoNed in the regulation of transcdlular water/fluid secretion by the pancreas. In particular, AQP1 expression appears to be infuenced by SEC. However, if SEC-responsive fluid secretion by PDCs is regulated by AQPs, it does not seem to be acutely controlled by protein expressmn.
vector
R~ASI0
P~SS2
RegSS3
M2206
S
Effects of Diet and GI Hormones on CRHSP-28 Phosphorylation and Subcellular Localization in Rat Pancreas gala M. Kaspar, Diana D. Thomas, Ning Weng, Guy E, Groblewski
UJ
CRHSP-28 is a Ca ++-sensitive regulatory protein that modulates the secretion of digestive enzymes from pancreatic acinar cells. To better understand the mechanisms by Which CRI-ISP-28 regulates secretion in response to diet and GI hormones, pbosphorylation of the protein was analyzed in isolated acini and in vivo. lsoelectric focusing and immunoblotting were used to detect CRHSP-28 phosphorylation. CCK stinMated CRHSP-28 pbosphorylation in isolated acini within 2 rain and over a range of physiological concemranons. Treatment with JMV-180, an agonist for the high affinity-state of the CCK-A receptor, confirmed the physiological relevance of the response. The effect of CCK was absolutely contingent on elevated Ca ++ as phosphorylation was blocked by" the intracenular Ca ++ buffer BAPTA, stimulated by Ca+ + ionophores, and unchanged by protein kinase C, -A, or -G activation. IV infusion of rats with secretory (0.1 ixg/kg/hr) and super-physiological (10 ~g/kg/hf)
Ill
E 1/1 os I11 l,v
AGA
R~IAS=
Am~as 1023(0,048) 0,825(0,060)* 1,107(0,171) 1,410(0,102)* 1,415(0.129)* e PDX-I 1.006(0,048) 1,758(0263)* 2,112(0.203)** 1145(0271) 0,767(0.058)* ~ulin 1,006(0,065) 2,004(0,199)* 2.422(0267)* 128(0.255) 1,09(0.172) Mean(SEM), *P< 0.05, ** P< 0.01, vs celt line expressingemptyvector
Abstracts
CTL
1i
~k
2tt
A-436
Sigmficant differences were not tound m the biophysical properties of PP responsive vs nonresponsive neurons. These data suggest that pancreatic vagal circuits controlling exocrine secretion undergo both short-term (caemlein treatment) and long-term (copper deficient diet) remodeling. Supported by NIH DK55530 and NSF 9816662.
Reduced islet regeneration alter aUoxan treatment in gastrin-CCK and gastrinTGFalpha deficient mice kennart Friis-Hansen, Troels Bock Aim: Duct to islet cell differentiation is thought to be regulated by growth factors. During pancreatic regeneration Mlowing duct ligation m rats the regenerating epithelium expresses gastrin and transforming growth factor-alpha (TGFalpha) mRNA and peptides. To examine the physiological importance of events we examined islet regeneration following alloxan treatment in wild type, gastriu deficient, gastrin-cek deficient, and gastrin TGFalpha deficient mice. Method: 20 12 weeks old mice fi'om each of the four groups of mice were give alloxan 250 mg/kg intra peritoneally. Only mice that 2 days after alloxan treatment had blood glucose above 14 mmoH were included in the study. Measurement of blood glucose and oral glucose tolerance test (OGTT) were performed once weekly during the following 8 weeks and compared to non treated controls. Resuhs: There were no difterences in body weight, weight of total pancreas or beta cell mass between the four groups of mice. Following alloxan treatment all mice were equally diabetic evaluated by fasting blood glucose and OGTT but whereas the wild type normalized their OGTT over the following 8 weeks the knockout mice had impaired normalization of OGTT. This was also reflected reduced beta cef mass (see table). Conclusion: Lack of gastrin, gastrin-CCK or gastrin-TGFalpha does not affect the development of the pancreas or the islets. However, following afloxan treatment, gastrin deficient mice had reduced islet regeneratwe ability. This was further inhibited by the additional loss of either CCK or TGFalpha. This suggests that these hormones are important for regeneration of the islets. Future expemnents will have to determine if exogenous administration of the hormones can enhance islet regeneration.
M2202
Stimulation of Pancreatic Secretion by Non-Sulfated CCK-58 Gives Insight into Neurohormonal Regulation of the Pancreas Joseph R. Reeve Jr., Mitsuyoshi Yamamoto, Gary, M. Green Non-sulfated cbolecystcMmn-58 (CCK-58ns) has been shown to be present m porcine and canine intestine, This form ot cholecystokinin may have been ignored by our laboratory in previous studies because it reacts very poorly in our radioimmunoassay with antibody 5135 (about 10% as potent as CCK-8s. However, it is present in the intestine at about half the concentration of CCK-58s. A maior bi-product from the synthesis and purification of rat CCK-58s is CCK-58ns. Syrabenc CCK-58ns was wall separated from CCK-58s, and was used to stinmlate pancreatic secretion in awake rats. CCK-58ns was as potent and one-half as effacions as CCK.-58s for stimulation ot pancreatic find and protein. CCK-8s was as potent as the CCK-58 peptides for stimulation of prutem, but CCK-8 did not stimulate fluid secretion (Figure). A model that would account tbr these results is one where both the acmar CCK-A receptor and the CCK-A receptor on intrapancreatic nerves regulate pancreatic secretion. The nanral CCK-A receptor is mediated by a neumtransmttter that can be blocked by atropine and it stinmlates both protein and fuid. We propose that this neural receptor is not activated by CCK-8s at any concetrations, possibly because CCK-8s is inactivated by neural endopeptidease before it reaches the neural CCK-A receptor. This is supported by the tact that CCK-58s is resistant to this endopepfidase degradation while CCK-8s is rapidly cleaved. We barther propose that the CCK-A receptor m~ aciuar cells is not activated by CCK-58ns and its inability to react with this receptor causes the decreased efficacy compared to CCK-58s. In conclusion, CCK-58ns is present in intestine and it has a unique pattern for stimulating pancreanc secretion that leads to new hypotheses about regulation of pancreatic secretion. -vllp- CC g 69 btS)
.-~
Wildtype
Gastdn-TGFalpha KO
Weight of pancreas (milltgrams) (day 0) Be~ cell ma~ (micrograms) {day 0) Futlng blood glucose
204~19
208~10
192:10
22125
773105
795143
752133
76789
7,70,5
8,5,10
10.8:0,9
123,0,7
W ~ t of pan~'tn~ (milli-
243,30
23414
26715
227~t4
cell mass (micrograms) (8 weeks)
690 78
584,82
370~61
442~60
(rmol/I) (8 weeks)
CO Ka (s)
Gastrln KO Gu~n.CCK
M2205
Pancreatic Reg Is a Modulator of Acinar Cell Differentiation: Influence on Cellular Lineage Didier Sanchez, Catherine Muller, Michael Zeniboan
Minutes
BACKGROUND & AIMS: Type I Reg gent, an acinar cell product, has been implicated in islet growth and maiutainence. Since the role of this acinar product in formation of islets is unclear, we tested if levels of reg l gent expression would influence acinar cellular differentiation. METHODS: We generated clones of AR42j cells, an amphicrine rat acinar pancreatic tumor line which expresses reg 1. We transfected it with a plasmid containing the entire coding sequence driven by a CMV promoter, and isolated stable clones with cDNA in sense (SS) or antisense (AS) orientation. Western blotting and Real Time-PCR were applied. RESULTS: Increased or reduced expression of Reg I mRNA and protein were confirmed. Morphologic analysis cells demonstrated more acinar-like differentiation in SS and a less differentiated state in AS. Amylase mRNA and protein levels increased in SS cells, whereas not protein levels changed in AS excepted mRNA levels decreased. AS cells showed increased cytokeratin proteins (CK-7 and 19), PDX-1 and insulin mRNAs. Conversely cytokeratin and PDX-1 decreased in SS cells (Table) CONCLUSIONS: These data demonstrated that reg I over-expression is linked to aciuar cell differentiation, and inhibition of reg l leads to the same acinar cells expressing ductal and [betal-cdl properties. We postulate that reg expression in acinar cells is important in maintaining pancreatic cell lineage, and as it changes, cells can dedifferentiate into ductal and/or [betal-cells. Relative expression of mRNAs.
M2203
Secretin (SEC) Regulates Expression of Aquaporins (AQP)In Isolated Pancreatic Duct Cells (PDCs) Carlos Mvarez, Christopher Waskewich The human pancreas, specifically duct cells, makes 1L of fluid daily in response to SEC. This process is not well defined at the cellular level Fluid secretion across epithelia is generally considered a passive, paracdlular phenomenon generated by osmotic gradients, but the recent discovery of AQPs, a differentially" expressed family of water channels, suggests that fluid secretion may be a well-regulated process in tissues like the b~ncreas with high secretory rates. We sought to define the pattern of AQP expression in PDCs and hypothesized that this would be under SEC control. METHODS: Segments of bovine main PD were harvested flesh, and maintained in explant cuhure lot 72h in basal media, The explants were tben exposed to SEC (0, 10 or 100nM). Epithelial cells were scraped off the PD connective tissue, lysed, and their protein and mRNA extracted. Western blot analysis was performed using antibodies against AQPI, 2, 4 and 8. RT-PCR was performed using primers for AQP1, with GAPDH used as housekeeper. RESULTS: AQP 1,4 and 8 protein was present in cultured duct cells, while AQP2 was not observed in the presence or absence of SEC. SEC stimulation did not aff?ct AQP protein levels at 24h after stimulation. AQP 1 RNA levels were initially depressed with exposure to SEC but recovered to levels above baseline (see char0. CONCLUSIONS: The pancreatic duct epithelium expresses an assortmem of AQPs that are likely invoNed in the regulation of transcdlular water/fluid secretion by the pancreas. In particular, AQP1 expression appears to be infuenced by SEC. However, if SEC-responsive fluid secretion by PDCs is regulated by AQPs, it does not seem to be acutely controlled by protein expressmn.
vector
R~ASI0
P~SS2
RegSS3
M2206
S
Effects of Diet and GI Hormones on CRHSP-28 Phosphorylation and Subcellular Localization in Rat Pancreas gala M. Kaspar, Diana D. Thomas, Ning Weng, Guy E, Groblewski
UJ
CRHSP-28 is a Ca ++-sensitive regulatory protein that modulates the secretion of digestive enzymes from pancreatic acinar cells. To better understand the mechanisms by Which CRI-ISP-28 regulates secretion in response to diet and GI hormones, pbosphorylation of the protein was analyzed in isolated acini and in vivo. lsoelectric focusing and immunoblotting were used to detect CRHSP-28 phosphorylation. CCK stinMated CRHSP-28 pbosphorylation in isolated acini within 2 rain and over a range of physiological concemranons. Treatment with JMV-180, an agonist for the high affinity-state of the CCK-A receptor, confirmed the physiological relevance of the response. The effect of CCK was absolutely contingent on elevated Ca ++ as phosphorylation was blocked by" the intracenular Ca ++ buffer BAPTA, stimulated by Ca+ + ionophores, and unchanged by protein kinase C, -A, or -G activation. IV infusion of rats with secretory (0.1 ixg/kg/hr) and super-physiological (10 ~g/kg/hf)
Ill
E 1/1 os I11 l,v
AGA
R~IAS=
Am~as 1023(0,048) 0,825(0,060)* 1,107(0,171) 1,410(0,102)* 1,415(0.129)* e PDX-I 1.006(0,048) 1,758(0263)* 2,112(0.203)** 1145(0271) 0,767(0.058)* ~ulin 1,006(0,065) 2,004(0,199)* 2.422(0267)* 128(0.255) 1,09(0.172) Mean(SEM), *P< 0.05, ** P< 0.01, vs celt line expressingemptyvector
Abstracts
CTL
1i
~k
2tt
A-436