help button home button Endocrine Society Molecular Endocrinology ENDO 08 Sessions Library
HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS
Molecular Endocrinology, doi:10.1210/me.2008-0255
This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow Supplemental Data
Right arrow A retraction has been published
Right arrow Submit a related Letter to the Editor
Right arrow Alert me when this article is cited
Right arrow Alert me when eLetters are posted
Right arrow Alert me if a correction is posted
Right arrow Citation Map
Services
Right arrow Email this article to a friend
Right arrow Similar articles in this journal
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Right arrow Reprints, Permissions and Rights
Citing Articles
Right arrow Citing Articles via HighWire
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Lavine, J. A.
Right arrow Articles by Attie, A. D.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Lavine, J. A.
Right arrow Articles by Attie, A. D.
Molecular Endocrinology 22 (12): 2716-2728
Copyright © 2008 by The Endocrine Society

Overexpression of Pre-Pro-Cholecystokinin Stimulates β-Cell Proliferation in Mouse and Human Islets with Retention of Islet Function

Jeremy A. Lavine1, Philipp W. Raess1, Dawn Belt Davis, Mary E. Rabaglia, Brent K. Presley, Mark P. Keller, Margery C. Beinfeld, Alan S. Kopin, Christopher B. Newgard and Alan D. Attie

Department of Biochemistry (J.A.L., P.W.R., D.B.D., M.E.R., M.P.K., A.D.A.), and Department of Medicine (D.B.D.), University of Wisconsin-Madison, Madison, Wisconsin 53706; Sarah W. Stedman Nutrition and Metabolism Center and Department of Pharmacology and Cancer Biology (B.K.P., C.B.N.), Duke University Medical Center, Durham, North Carolina 27704; and Department of Pharmacology (M.C.B.), Tufts University, and Molecular Pharmacology Research Center (A.S.K.), Tufts Medical Center, Medford, Massachusetts 02111

Address all correspondence and requests for reprints to: Alan D. Attie, 433 Babcock Drive, Room 543A, Madison, Wisconsin 53706. E-mail: adattie{at}wisc.edu.


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
Type 1 and type 2 diabetes result from a deficit in insulin production and β-cell mass. Methods to expand β-cell mass are under intensive investigation for the treatment of type 1 and type 2 diabetes. We tested the hypothesis that cholecystokinin (CCK) can promote β-cell proliferation. We treated isolated mouse and human islets with an adenovirus containing the CCK cDNA (AdCMV-CCK). We measured [3H]thymidine and BrdU incorporation into DNA and additionally, performed flow cytometry analysis to determine whether CCK overexpression stimulates β-cell proliferation. We studied islet function by measuring glucose-stimulated insulin secretion and investigated the cell cycle regulation of proliferating β-cells by quantitative RT-PCR and Western blot analysis. Overexpression of CCK stimulated [3H]thymidine incorporation into DNA 5.0-fold and 15.8-fold in mouse and human islets, respectively. AdCMV-CCK treatment also stimulated BrdU incorporation into DNA 10-fold and 21-fold in mouse and human β-cells, respectively. Glucose-stimulated insulin secretion was unaffected by CCK expression. Analysis of cyclin and cdk mRNA and protein abundance revealed that CCK overexpression increased cyclin A, cyclin B, cyclin E, cdk1, and cdk2 with no change in cyclin D1, cyclin D2, cyclin D3, cdk4, or cdk6 in mouse and human islets. Additionally, AdCMV-CCK treatment of CCK receptor knockout and wild-type mice resulted in equal [3H]thymidine incorporation. CCK is a β-cell proliferative factor that is effective in both mouse and human islets. CCK triggers β-cell proliferation without disrupting islet function, up-regulates a distinct set of cell cycle regulators in islets, and signals independently of the CCK receptors.


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
TYPE 1 AND TYPE 2 diabetes result from inadequate β-cell mass. In type 1 diabetes, autoimmune destruction of pancreatic β-cells results in a complete loss of insulin production. Islet transplantation has recently been investigated as a potential cure for type 1 diabetes. Unfortunately, multiple cadaveric donors are typically required to amass sufficient islet numbers to restore euglycemia (1, 2, 3). In patients with type 2 diabetes, pancreatic insulin production cannot compensate for insulin resistance due to inadequate functional β-cell mass. Methods to expand β-cell mass therefore have potential therapeutic applications for type 1 and type 2 diabetes.

Several growth factors have been identified that can stimulate rodent β-cell proliferation. These mitogens include hepatocyte growth factor (HGF) (4, 5, 6), glucagon-like peptide 1 (GLP-1) (7, 8), glucose-dependent insulinotropic peptide (9), placental lactogen (10), PTHrP (11), and trefoil factor 3 (TFF3) (12). Of these factors, only HGF (5, 6) and TFF3 (12) have been shown to also stimulate human β-cell proliferation, although in each case, with less efficiency than in rodent islets.

Cholecystokinin (CCK) has been extensively studied as a gastrointestinal hormone and neuropeptide. In the gastrointestinal tract, CCK is secreted from duodenal I-cells in response to fat and protein and stimulates pancreatic acinar secretion and gall bladder contraction. In the central nervous system, CCK modulates behavioral functions including satiety, anxiety, and memory. Two recent studies have suggested a role for CCK in islet cell proliferation. Kuntz et al. (13) demonstrated that 8 d of injection of the mature eight-amino acid product of the CCK gene, CCK8, after streptozotocin treatment in rats resulted in reduced hyperglycemia, increased plasma insulin, and increased β-cell proliferation. Additionally, Chen et al. (14) showed that CCK8 treatment of partially pancreatectomized rats stimulated islet cell proliferation.

We previously reported that CCK mRNA expression is dramatically up-regulated in pancreatic islets of the ob/ob mouse, a model of insulin resistance and compensatory expansion of β-cell mass (15). Given this finding and previous work linking CCK to islet cell proliferation, we hypothesized that CCK promotes β-cell proliferation. To test this hypothesis, we constructed an adenovirus containing the CCK cDNA. We report here that overexpression of CCK in isolated mouse and human islets potently stimulates β-cell proliferation with retention of islet function. Additionally, we demonstrate that CCK-induced β-cell proliferation occurs without up-regulation of D-type cyclins. Instead, CCK increases the abundance of cyclins A, B, E, cdk1, and cdk2, a pattern similar to that recently reported for β-cell proliferation mediated by the homeodomain transcription factor Nkx6.1 (16), but distinct from other islet proliferative factors.


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
AdCMV-CCK Treatment of Islets Produces CCK mRNA and Peptides
To overexpress cholecystokinin in isolated islets, we constructed an adenovirus containing the mouse cholecystokinin (CCK) cDNA encoding the pre-pro-cholecystokinin protein, driven by the cytomegalovirus (CMV) promoter (AdCMV-CCK). Quantitative RT-PCR analysis of AdCMV-β-Gal control and AdCMV-CCK treated isolated mouse islets demonstrated increased CCK mRNA expression. Because CCK is expressed at nearly undetectable levels in lean mouse islets, AdCMV-CCK treatment resulted in an approximate 20,000-fold induction over control islets (Fig. 1AGo, P < 0.001). We performed HPLC fractionation followed by RIA to determine whether AdCMV-CCK treatment resulted in production of CCK peptides. Using sulfated CCK amide standards, we detected high levels of CCK8 and CCK12 amides, and low levels of CCK22 and other unidentified CCK species in AdCMV-CCK-treated islets (Fig. 1BGo). Overall, we found 370 pg of total CCK amides per µg of total protein. No CCK peptides were detected in AdCMV-β-Gal-treated islets.


Figure 1
View larger version (11K):
[in this window]
[in a new window]

  		Fig. 1. AdCMV-CCK Treatment Stimulates Production of CCK mRNA and Peptides

A, CCK mRNA expression, normalized to β-actin, in AdCMV-β-Gal- and AdCMV-CCK-treated islets by Taqman quantitative RT-PCR (P < 0.001). B, CCK amide species in AdCMV-CCK-treated islets. HPLC fractionation followed by CCK RIA analysis was performed. Peaks were identified by CCK amide standards.

 
Treatment of Mouse and Human Islets with AdCMV-CCK Stimulates β-Cell Proliferation with Retention of Islet Function
To examine the proliferative effect of CCK overexpression, we measured [3H]thymidine incorporation into DNA in AdCMV-CCK-treated mouse islets vs. control AdCMV-β-Gal-treated and untreated islets. Mouse islets treated with AdCMV-CCK exhibited a 5-fold increase in incorporation of [3H]thymidine vs. control islets (Fig. 2AGo; P < 0.001). Moreover, we found that CCK overexpression stimulated a 4-fold and 65-fold increase in the mRNA abundance of the proliferative markers PCNA and Ki67, respectively, by quantitative RT-PCR (P < 0.01 for both; data not shown). The potential therapeutic applications of our findings prompted us to explore whether we could show similar effects in human islets. In islets from 13 independent human donors, [3H]thymidine incorporation into DNA was stimulated by an average of 15.8-fold in response to AdCMV-CCK treatment compared with AdCMV-β-Gal-treated control islets (Fig. 3AGo; P < 0.001). Similarly to mouse islets, we found that CCK overexpression in human islets stimulated a 5-fold and 17-fold increase in the mRNA abundance of the proliferative markers PCNA and Ki67, respectively (P < 0.01 for both; data not shown).


Figure 2
View larger version (38K):
[in this window]
[in a new window]

  		Fig. 2. AdCMV-CCK Treatment Stimulates Mouse β-Cell Proliferation

A, [3H]thymidine incorporation assay on untreated (NT), AdCMV-β-Gal-treated (β-gal), and AdCMV-CCK (CCK)-treated islets. Each point represents an individual islet preparation treated as indicated. B, Glucose-stimulated insulin secretion assay on untreated, AdCMV-β-Gal-, and AdCMV-CCK-treated islets. *, P < 0.05 for AdCMV-CCK vs. AdCMV-β-Gal and untreated islets under basal conditions (n = 5). C and D, Representative islet sections of AdCMV-β-Gal- and AdCMV-CCK-treated islets. BrdU is stained green, insulin is stained red, and 4',6-diamidino-2-phenylindole (DAPI) is stained blue. E, Quantitation of islet sections from three independent adenoviral treatments. β-Cells were BrdU positive if BrdU staining was overlaid with DAPI staining, and the nuclei were surrounded by three sides of insulin staining. F and G, Representative images of flow cytometry analysis in AdCMV-β-Gal- and AdCMV-CCK-treated islets. Cell cycle phases are indicated on the x-axis. H, Quantitation of three individual islet treatment experiments analyzed by flow cytometry analysis. *, P < 0.05 for AdCMV-CCK vs. AdCMV-β-Gal at the indicated cell cycle phases.

 

Figure 3
View larger version (38K):
[in this window]
[in a new window]

  		Fig. 3. AdCMV-CCK Treatment Stimulates Human β-Cell Proliferation

A, [3H]thymidine incorporation assay on untreated (NT), AdCMV-β-Gal-treated (β-gal), and AdCMV-CCK (CCK)-treated islets. Each point represents an individual islet donor treated as indicated. B, Glucose-stimulated insulin secretion assay on untreated, AdCMV-β-Gal-, and AdCMV-CCK-treated islets. No significant differences were found (n = 7). C and D, Representative islet sections of AdCMV-β-Gal- and AdCMV-CCK-treated islets. BrdU is stained green, insulin is stained blue, and glucagon is stained red. E, Quantitation of islet sections from three independent adenoviral treatments. β-Cells or {alpha}-cells were BrdU positive if BrdU staining was surrounded by three sides of insulin or glucagon staining. Yellow and white arrows indicate representative proliferating {alpha}-cells and β-cells, respectively. F and G, Representative images of flow cytometry analysis in AdCMV-β-Gal- and AdCMV-CCK-treated islets. Cell cycle phases are indicated on the x-axis. H, Quantitation of four individual islet treatment experiments analyzed by flow cytometry analysis. *, P < 0.05 for AdCMV-CCK vs. AdCMV-β-Gal at the indicated cell cycle phases.

 
Growth factor- or oncogene-induced islet cell proliferation has been previously shown to result in a decrease in key islet functions (17). We measured glucose-stimulated insulin secretion in AdCMV-CCK-treated mouse and human islets vs. control AdCMV-β-Gal-treated and untreated islets, as a measure of β-cell function. In mouse islets, overexpression of CCK caused a 3.9-fold (P < 0.05) and 2.4-fold (P = 0.09) increase in the percentage of total insulin secreted under basal low-glucose and stimulated high-glucose conditions, respectively, with no change in insulin content (Fig. 2BGo). When comparing the amount of insulin secreted relative to basal conditions, no significant changes were observed between AdCMV-CCK-treated and control islets. In human islets, AdCMV-CCK treatment had no significant effect on insulin secretion at basal low glucose, stimulated high glucose, or fold vs. basal conditions (Fig. 3BGo). Therefore, AdCMV-CCK treatment had no detrimental effect on glucose-stimulated insulin secretion in mouse or human islets.

To determine the islet cell type responsible for the increased incorporation of [3H]thymidine into DNA, we stained isolated islets treated with either AdCMV-β-Gal or AdCMV-CCK for 5-bromo-2'-deoxyuridine (BrdU) and insulin. In control mouse islets, BrdU incorporation into β-cells was a relatively rare event, occurring approximately once in every five islet sections (Fig. 2Go, C and E). In AdCMV-CCK-treated mouse islets, we observed that, on average, two β-cells per islet section incorporated BrdU (Fig. 2Go, D and E). Overall, CCK overexpression increased mouse β-cell BrdU incorporation 10-fold vs. control islets (Fig. 2EGo; P < 0.001). When we performed similar initial studies in human islets, we found that approximately half of AdCMV-CCK-treated islet cells that incorporated BrdU did not costain for insulin. Therefore, we stained for glucagon and insulin, as well as BrdU. In control human islets, incorporation of BrdU into any cell type was extremely rare, occurring once every 10 islet sections (Fig. 3Go, C and E). In AdCMV-CCK-treated human islets, however, we observed that two to three {alpha}-cells and two to three β-cells incorporated BrdU per islet section (Fig. 3Go, D and E). These data translated into 21-fold (P < 0.05) and 32-fold (P < 0.01) increases in {alpha}-cell and β-cell BrdU incorporation, respectively (Fig. 3EGo). Of all islet cells that incorporated BrdU in AdCMV-CCK-treated human islets, 91% of cells were clearly identified as {alpha}-cells or β-cells.

[3H]thymidine and BrdU incorporation assays are only measures of DNA synthesis, and thus our results could reflect DNA damage repair rather than cell replication. We therefore performed flow cytometric analysis of dispersed mouse and human islet cells to confirm that AdCMV-CCK treatment causes progression through all phases of the cell cycle. Using insulin immunostaining, we determined the proportion of mouse and human β-cells at each stage of the cell cycle. CCK overexpression stimulated a 12.7- and 2.5-fold increase in the number of β-cells in S phase, in mouse (Fig. 2HGo and Table 1Go; P < 0.05) and human (Fig. 3HGo and Table 2Go, P < 0.01) islets, respectively. AdCMV-CCK treatment also caused a 7.1% and 7.3% decrease in the number of β-cells in G0 or G1 phase in mouse (Fig. 2HGo and Table 1Go; P < 0.05) and human (Fig. 3HGo and Table 2Go; P < 0.05) islets, respectively. Finally, overexpression of CCK resulted in an 8.0% and 32.6% increase in the number of G2 or M phase β-cells in mouse (Fig. 2HGo and Table 1Go; P = 0.11) and human (Fig. 3HGo and Table 2Go; P < 0.05) islets, respectively. Therefore, CCK overexpression stimulated β-cells to progress through all phases of the cell cycle. We show using multiple lines of evidence that AdCMV-CCK treatment increased mouse and human β-cell proliferation with retention of β-cell function.


View this table:
[in this window]
[in a new window]

  		Table 1. β-Cell Flow Cytometry Analysis of Mouse Islets Treated with AdCMV-β-Gal or AdCMV-CCK

 

View this table:
[in this window]
[in a new window]

  		Table 2. β-Cell Flow Cytometry Analysis of Human Islets Treated with AdCMV-β-Gal or AdCMV-CCK

 
AdCMV-CCK Treatment Up-Regulates a Specific Pattern of Cyclins and cdks
Cell cycle regulation in islet biology is a relatively young field. Some reports have investigated the mechanism of islet cell cycle regulation in rodent models, but very few have confirmed these results in a human islet model. We therefore investigated the impact of CCK overexpression on genes involved in cell cycle control by quantitative RT-PCR and Western blot analysis.

In mouse islets, AdCMV-CCK treatment strongly induced the mRNA expression of cyclins A2 (32.0-fold), B1 (13.8-fold), B2 (20.0-fold), E1 (25.6-fold), and E2 (20.8-fold) vs. control islets (Fig. 4AGo; P < 0.01 for all). We confirmed that cyclin A and B proteins are at low abundance in control islets and significantly induced by CCK overexpression (Fig. 4CGo). Cyclin E protein increased only 1.2-fold in CCK overexpressing islets (Fig. 4CGo; P < 0.05), despite the 20- to 25-fold increase in its mRNA abundance (Fig. 4AGo). Surprisingly, AdCMV-CCK did not significantly induce the mRNA expression of cyclins D1, D2, or D3 (Fig. 4AGo). Because D-type cyclins, specifically cyclin D2, can be regulated at the level of translation (18), we confirmed that abundance of cyclin D1, D2, and D3 protein is also not significantly changed by CCK overexpression (Fig. 4CGo).


Figure 4
View larger version (20K):
[in this window]
[in a new window]

  		Fig. 4. AdCMV-CCK Treatment of Mouse Islets Up-Regulates a Novel Set of Cyclin and cdk Molecules

A and B, Quantitative RT-PCR analysis of cyclin and cdk mRNA abundance in mouse islets. Cyclin and cdk mRNA abundance were normalized to β-actin for AdCMV-β-Gal- and AdCMV-CCK-treated islet cDNA. Fold change vs. AdCMV-β-Gal was calculated for each pair of treated islets. and an average fold was calculated (n = 3). *, P < 0.05; **, P < 0.01. C, Western blot analyis of cyclin protein abundance. Cyclin protein abundance was quantitated by densitometry and normalized to β-tubulin. β-Gal and CCK 1–3 indicate three independent experiments. Fold change was determined for each individual experiment and averaged. *, P < 0.05; ***, quantitation was not possible because no protein was detectable in AdCMV-β-Gal-treated islets.

 
We also analyzed the cell cycle in human islets, including more detailed studies of the time course of proliferation and cell cycle gene expression. We found that CCK overexpression led to modestly increased [3H]thymidine incorporation at 18 h post treatment (2.0 fold, P = 0.07; Fig. 5AGo), but at 42 h post treatment we observed equal stimulation of [3H]thymidine incorporation (15.4-fold, P < 0.01; Fig. 5BGo) compared with 66 h post treatment (Fig. 3Go). At 66 h post treatment, we found that CCK overexpression led to a similar pattern of human cell cycle gene expression as was observed in mouse islets. CCK overexpression stimulated the mRNA expression of cyclins A1 (2.7-fold), A2 (31.2-fold), B1 (4.2-fold), B2 (10.1-fold), E1 (32.5-fold), and E2 (32.0-fold) in human islets (Fig. 5GGo; P < 0.01 for all). At the protein level, we found that cyclins A and B are dramatically induced, whereas cyclin E was only up-regulated 2.0-fold by CCK overexpression (Fig. 5IGo; P < 0.05). We found that at 18 h post treatment, rather surprisingly, CCK overexpression had already significantly increased the expression of cyclins A2 (13.0-fold; P < 0.05), B1 (5.8-fold; P < 0.01), B2 (10.5-fold; P < 0.05), E1 (31.3-fold; P < 0.01), and E2 (51.5-fold; P < 0.05). This pattern of gene expression was maintained at 42 h post treatment as well (Fig. 5EGo). With respect to D-type cyclins, CCK overexpression did not consistently increase or decrease cyclins D1, D2, or D3 mRNA more than 2-fold at any time point (Fig. 5Go, C, E, and G). At the protein level, cyclin D1 was down-regulated by 50%, whereas cyclin D3 was unchanged at 66 h post treatment (Fig. 5IGo). Cyclin D2 protein was surprisingly undetectable in proliferating human islets, despite the use of four different antisera.


Figure 5
View larger version (22K):
[in this window]
[in a new window]

  		Fig. 5. AdCMV-CCK Treatment of Human Islets Up-Regulates a Novel Set of Cyclin and cdk Molecules

A and B, [3H]thymidine incorporation assays at the indicated time points post treatment. C–H, Quantitative RT-PCR analysis of cyclin and cdk mRNA abundance in human islets at the indicated time points post treatment. Abundance of cyclin and cdk mRNA was normalized to β-actin for AdCMV-β-Gal- and AdCMV-CCK-treated islet cDNA. Fold change vs. AdCMV-β-Gal was calculated for each islet donor, and an average fold was calculated (n = 3 for C–F; n = 5 for G and H). *, P < 0.05; **, P < 0.01. I, Western blot analysis of cyclin protein abundance. Cyclin protein abundance was quantitated by densitometry and normalized to β-tubulin. β-Gal and CCK 1–3 indicate three independent experimental donors. Fold change was determined for each individual experiment and averaged. *, P < 0.05; ***, quantitation was not possible because no protein was detectable in AdCMV-β-Gal-treated islets.

 
Cyclin F is a protein regulated similarly to A-type and B-type cyclins in the cell cycle, but its exact function is unclear (19). Interestingly, CCK overexpression significantly increased cyclin F mRNA expression by 11.4-fold in mouse islets (Fig. 4AGo; P < 0.01) and 3.1-fold in human islets at 66 h post treatment (Fig. 5GGo; P < 0.01). These studies indicate that overexpression of CCK in mouse and human islets can stimulate β-cell proliferation without increased abundance of D-type cyclins and that these cell cycle effects precede proliferation in human islets.

We also investigated the mRNA expression and protein abundance of cdk molecules. In mouse islets, we found that cdk1 and cdk2 mRNA were up-regulated 15.8-fold (P < 0.05) and 7.5-fold (P < 0.01), respectively, by CCK overexpression (Fig. 4BGo). We confirmed that cdk1 and cdk2 were also increased at the protein level (data not shown). No change was observed in cdk4 mRNA expression (Fig. 4BGo) or cdk4 protein abundance (data not shown). Interestingly, we found that CCK overexpression significantly increased cdk6 mRNA abundance (Fig. 4BGo; P < 0.05). However, in accordance with previous studies (20), we found that cdk6 protein is not detectable in mouse islets (data not shown).

In human islets, we found that cdk1 and cdk2 mRNA were up-regulated 20.2-fold and 6.6-fold in AdCMV-CCK-treated islets at 66 h post treatment (Fig. 5HGo; P < 0.01 for both). At 18 h post treatment, cdk1 and cdk2 mRNA abundance was already increased to 17.4-fold (P < 0.05) and 6.2-fold (P < 0.01), respectively (Fig. 5DGo). We confirmed that cdk1 and cdk2 proteins were increased in AdCMV-CCK-treated islets at 66 h post treatment (data not shown). Similarly to mouse islets, no consistent expression changes greater than 2-fold were detected at the mRNA or protein level for cdk4 or cdk6 molecules (Fig. 5Go, D, F, and H and data not shown).

We have demonstrated that A-type, B-type, and E-type cyclins, along with their binding partners cdk1 and cdk2, are up-regulated during adult human and mouse β-cell proliferation. Additionally, these gene expression changes precede AdCMV-CCK-mediated increases in β-cell proliferation.

AdCMV-CCK Treatment Stimulates β-Cell Proliferation Independent of the CCK1 and CCK2 Receptor
To test which CCK receptor was responsible for CCK-triggered β-cell proliferation, we measured [3H]thymidine incorporation into DNA of islets from CCK receptor-deficient mice or in human islets in the presence of pharmacological inhibitors. In islets isolated from mice null for both the CCK1 and CCK2 receptor (21, 22), AdCMV-CCK treatment stimulated [3H]thymidine incorporation into DNA 4.3-fold vs. contol islets (Fig. 6AGo; P < 0.01). In the presence of devazepide (a CCK1 receptor antagonist) and YM022 (a CCK2 receptor antagonist), human islets treated with AdCMV-CCK incorporated [3H]thymidine into DNA equally well as in vehicle-treated human islets (Fig. 6BGo). These two results suggest that AdCMV-CCK treatment of human or mouse islets stimulates β-cell proliferation through a pathway independent of either known CCK receptor.


Figure 6
View larger version (13K):
[in this window]
[in a new window]

  		Fig. 6. CCK Overexpression Stimulates β-Cell Proliferation Independent of the Two CCK Receptors

A, [3H]thymidine incorporation assays of AdCMV-β-Gal- and AdCMV-CCK-treated islets in wild-type (gray bars) and CCK1 and CCK2 receptor double knockout mice (black bars) (n = 3). Error bars represent mean ± SEM. B, [3H]thymidine incorporation assays of AdCMV-β-Gal- (gray bars) and AdCMV-CCK- (black bars) treated human islets. Islets were treated with either vehicle (0.1% ethanol) or CCK1 and CCK2 receptor antagonists devazepide and YM022, respectively, for 48 h (n = 5). Error bars represent mean ± SEM. KO, Knockout.

 

   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
MATERIALS AND METHODS
 REFERENCES
 
Insufficient β-cell mass and insulin production are defining features of both type 1 and type 2 diabetes (23, 24, 25). Expansion of islet mass, either ex vivo before transplantation or in vivo to compensate for increased insulin resistance, are two possible therapeutic interventions to treat individuals with either form of diabetes (17). In the present work, we report that overexpression of CCK stimulates robust β-cell proliferation in mouse and human islets while retaining β-cell function. Additionally, AdCMV-CCK treatment up-regulates cyclins A, B, E, cdk1, and cdk2 without increasing D-type cyclin abundance.

Previous studies have identified other mouse and human β-cell mitogens. In rodents, β-cell peptide growth factors include HGF (4, 5, 6), GLP-1 (7, 8), glucose-dependent insulinotropic peptide (9), placental lactogen (10), PTHrP (11), and TFF3 (12), but these factors are less effective in human islets than in rodent islets. In human islets, overexpression of intracellular signaling pathway components or transcription factors, including constitutively active Akt (26), protein kinase C-{zeta} (11), p8 (27), mouse pax4 (28), and Nkx6.1 (16), stimulates human islet or β-cell proliferation. Two peptide growth factors, TFF3 (12) and HGF (5, 6), also trigger human islet cell proliferation. By three independent methodologies, we demonstrate that overexpression of CCK can stimulate β-cell proliferation in mouse islets (Fig. 2Go and Table 1Go). Strikingly, in human islets CCK overexpression can stimulate a robust 15- to 20-fold increase in β-cell proliferation (Fig. 3Go and Table 2Go). In human β-cells, these large increases are likely due to the very low capacity of human β-cells to replicate in culture. CCK can now be added to this short list of peptide growth factors in rodent and human β-cells.

In AdCMV-CCK-treated human islets, 45% of proliferating cells costain for insulin, and 45% costain for glucagon. Although 80% of all mouse islet cells that incorporated BrdU were β-cells, {alpha}-cell proliferation was not specifically addressed and cannot be excluded. To our knowledge, this is the first report of a growth factor that was overexpressed under the control of a CMV promoter that can stimulate both {alpha}-cell and β-cell proliferation. For example, two recent studies involving CMV promoter-driven overexpression of Nkx6.1 or TFF3 in rat islets established that the increase in islet cell replication occurred almost exclusively in β-cells in both cases (12, 16). In the setting of human islet cell transplantation, {alpha}-cell proliferation is a finding with potentially important therapeutic implications. The major clinical criterion for islet cell transplantation therapy is severe hypoglycemia. A therapy that causes expansion of β-cells in the absence of {alpha}-cell proliferation could place the patient at risk for recurrent hypoglycemia, an outcome that can potentially be avoided with concomitant {alpha}-cell expansion. Therefore, coexpansion of {alpha}-cells and β-cells via CCK-dependent pathways could help to restore hypoglycemic responsiveness and achieve insulin independence in patients receiving islet transplants (29).

In order for expansion of β-cell mass to be therapeutic, it is crucial that islet function be maintained. Overexpression of CCK triggers β-cell proliferation with enhanced total insulin secretion at both basal and stimulated conditions in mouse islets (Fig. 2BGo) and without any significant changes in human islets (Fig. 3BGo). These experiments address the effect of CCK overexpression upon the insulin-secretory capacity of the individual β-cell because the data are normalized to total β-cell numbers, which removes the effect of CCK as a mitogen. Enhanced insulin secretion is not surprising because CCK has previously been shown to act as an incretin hormone. In rodents, physiological doses of CCK enhance insulin secretion in vivo (30) and in pancreatic perfusions (31). In humans, however, supraphysiological doses of CCK are necessary to stimulate insulin secretion (32). These species-specific differences may explain why CCK overexpression increased insulin secretion in mouse islets significantly without any significant changes in human islets.

Previous genetic effects on islet cell cycle regulation focus strongly on the D-type cyclins and the G0/G1 to S transition. Genetic studies on cdk4 include the cdk4-deficient mouse and the cdk4 knock-in mouse, which expresses an active cdk4 mutant that cannot be inhibited by p16ink4a. Mice deficient for cdk4 develop insulin-deficient diabetes due to reduced β-cell mass, whereas cdk4 knock-in mice demonstrate β-cell hyperplasia (33). Furthermore, cyclin D2-deficient mice display reduced β-cell mass and proliferation, leading to glucose intolerance and diabetes (34, 35). Additionally, cyclin D1 heterozygosity in the cyclin D2-deficient background results in life-threatening diabetes at an earlier age than cyclin D2 deficiency alone (34). Transgenic overexpression of cyclin D1 in the β-cell stimulates β-cell proliferation and hyperplasia (36). Moreover, overexpression of cyclin D1 and cdk4 can stimulate mouse and human β-cell proliferation in vitro (37). These genetic studies indicate an important role for cyclin D1, D2, and cdk4 in β-cell proliferation.

Mechanistic work on growth factor-stimulated β-cell proliferation supports the above genetic studies. Prolactin and GH stimulate β-cell proliferation by up-regulating cyclin D2 through a pathway dependent upon signal transducer and activator of transcription 5 (STAT5) (38, 39). Increased Wnt signaling in vivo and in vitro increases β-cell proliferation by stimulating cyclin D2 expression (40). GLP-1 stimulates β-cell proliferation by inducing cyclin D1 expression through protein kinase A (PKA)-, phosphoinositol 3-kinase (PI3K)-, and MAPK-dependent pathways (9). Overexpression of constitutively active Akt in β-cells stimulates β-cell proliferation by up-regulating cyclin D1, D2, and cdk4 activity (18). These studies indicate that cyclin D1, D2, and cdk4 are critical for β-cell proliferation through PKA-, PI3K-, MAPK-, STAT5-, and Akt-dependent pathways. CCK signaling, however, stimulates β-cell proliferation without up-regulating cyclin D1, D2, or cdk4 (Figs. 4Go and 5Go) and may suggest that CCK signals independently of these pathways. In fact, we were unable to detect any cyclin D2 in proliferating human islets (Fig. 5Go). If human islets do not express cyclin D2 and cannot up-regulate its expression, then lack of cyclin D2 could explain the weak effects of rodent β-cell-proliferative factors on human islets.

Overexpression of CCK instead triggers β-cell proliferation similarly to the transcription factor Nkx6.1. CCK signaling increases the mRNA and protein abundance of cyclin A, B, E, cdk1, and cdk2 (Figs. 4Go and 5Go). In fact, these changes in gene expression precede β-cell proliferation, suggesting they are causal and not reactive to the proliferative stimulus (Fig. 5Go). Adenoviral overexpression of Nkx6.1 in isolated rat islets stimulates β-cell proliferation by up-regulating the mRNA abundance of cyclin A1, B1, B2, E1, cdk1, and cdk2 (16). A time course study also revealed that induction of cyclin E by Nkx6.1 precedes the increase in expression in the other cyclins, a pattern different than what was observed in the current study with CCK (16). Moreover, cyclin E overexpression was sufficient to stimulate proliferation of rat islets (16). Although no change in cyclin D1 or D2 mRNA abundance was detected, Nkx6.1 overexpression increased cyclin D2 protein abundance slightly (16). Therefore, CCK overexpression stimulates β-cell proliferation by up-regulating a set of cell cycle genes that are similar, but not identical, to those elicited by Nkx6.1 expression, and clearly distinct from the pattern of growth factor-mediated up-regulation of D-type cyclins. Although Nkx6.1 mRNA abundance was unchanged in AdCMV-CCK-treated islets (data not shown), these data do not preclude the possibility that CCK signals to enhance endogenous Nkx6.1 activity to trigger β-cell proliferation.

This unique pattern of cyclin and cdk gene expression is not simply an artifact of in vitro adenoviral studies involving CCK or Nkx6.1. Previous work in our laboratory comparing the diabetes-resistant C57BL/6ob/ob and diabetes-susceptible BTBRob/ob mouse strains identified similar genes as differentially regulated at the mRNA level (41). C57BL/6ob/ob islets up-regulate cyclin A2, B1, and cdk1 transcripts, are diabetes-resistant, and show increased islet cell proliferation vs. lean controls (41). In contrast, BTBRob/ob islets do not up-regulate cyclin A2, B1, or cdk1 transcripts, are overtly diabetic, and show no increased islet cell proliferation vs. lean controls (41). The concordance between the pattern of cyclin and cdk expression we see in our type 2 diabetes model and in our CCK studies suggests that CCK could be signaling through pathways that normally defend animals from obesity-induced diabetes.

CCK receptor signaling shares many common downstream pathways with other β-cell proliferative factors. CCK receptors activate PI3K, Akt, Jak/STAT, MAPK, PKA, protein kinase C, and epidermal growth factor receptor pathways (42). Because of this significant overlap, we decided to test if CCK receptors were necessary for CCK-triggered β-cell proliferation. CCK receptors have previously been shown to colocalize with {alpha}- and β-cells (CCK1 receptor) and {delta}-cells (CCK2 receptor) (43). We found that islets from CCK receptor-deficient mice were as responsive as wild-type islets in response to CCK overexpression (Fig. 6AGo). Additionally, human islets treated with CCK receptor antagonists were also as responsive to CCK overexpression as vehicle-treated islets (Fig. 6BGo). Because the CCK receptor deletions and the CCK receptor antagonists affect all islet cells, we conclude that {alpha}-cell and β-cell proliferation is CCK receptor independent. We propose three possible explanations for this surprising finding. 1) CCK signals through a third yet-to-be identified CCK receptor; 2) overexpression of CCK stimulates another receptor pathway; 3) CCK signals independently of any receptor. Our data do not allow us to distinguish among any of these three possibilities.

In summary, we have identified a factor that stimulates β-cell proliferation with retention of β-cell insulin-secretory function in both mice and humans. CCK overexpression triggers β-cell proliferation through a distinct pathway from those previously described. The robust effect of CCK on human β-cell proliferation may have significant therapeutic potential in vivo in islet cell transplant or in the augmentation of functional β-cell mass. Understanding the signaling pathways that activate the members of the cell cycle machinery in response to CCK may also reveal additional therapeutic targets.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
REFERENCES
 
Reagents
Sulfated CCK8 amide was purchased from Neosystems (Strasbourg, France). Sulfated CCK12 and CCK12 were synthesized by Kouki Kitagawa at Niigata University of Pharmacy and Applied Life Sciences (Niigata, Japan). YM022 and Devazepide were purchased from Tocris Biosciences (Ellisville, MO) and dissolved in 100% ethanol at x1000 concentrations.

Adenovirus Construction
The mouse CCK cDNA (NM_031161) was cloned into the FF001 plasmid, a derivative of pACCMV.pLpA. Sequence-verified plasmid preparations were used to construct a recombinant adenovirus by cotransfecting pACCMV-CCK and pJM17, a plasmid containing the adenovirus genome lacking the E1A gene required for viral replication (44). The crude virus was amplified, purified by centrifugation on a CsCl gradient, and titered with an endpoint-limiting dilution assay.

Mice, Islet Isolations, Transduction, and Culture
Islets from 129/SvEv mice were used for [3H]thymidine, BrdU incorporation, and quantitative RT-PCR experiments. Islets from C57BL/6-CCKlacZ/lacZ mice (45), which are CCK deficient, were used for glucose-stimulated insulin secretion, flow cytometry, and Western blot experiments because of their larger islet yield. Mouse pancreatic islets were isolated by collagenase digestion (46). After handpicking, islets were washed twice in islet media: RPMI 1640 with 8 mM glucose, 10% FBS, and 1% antibiotic/antimycotic. Islets were then treated with AdCMV-CCK or AdCMV-β-Gal with an multiplicity of infection of approximately 250–500 (4.7 x 107 plaque-forming units/100–200 islets) for 18–20 h. Islets were moved daily to fresh islet media. Mouse procedures were approved by the Association for Assessment and Accreditation of Laboratory Animal Care to meet acceptable standards of humane animal care.

Human Islet Procedures
Human islets were obtained from participating islet cell resource centers. Islets were treated with AdCMV-CCK or AdCMV-β-Gal with a multiplicity of infection of approximately 25–50 (4.7 x 107 plaque-forming units/1000–2000 islet equivalent units) for 18–20 h. After viral treatment, media were changed to fresh CMRL 1066 (Cellgro, Manassas, VA) containing 2.5% human serum albumin. On d 2 post treatment, islets were washed three times in 8 mM glucose RPMI media supplemented with 10% FBS and 1% penicillin/streptomycin and cultured an additional 18–20 h. At this point, experiments were carried out identically to mouse islets. All protocols were approved by the Institutional Review Board at the University of Wisconsin-Madison.

Identification of CCK Forms
On d 4 post treatment, HPLC was carried out on flash-frozen mouse islet extracts with a Waters Alliance HPLC system (Waters Corp., Milford, MA) using a 4.6 x 250 Symmetry Shield RP 18 column. The islet cell extracts were made with cold 0.1 N HCl, the islets were sonicated, and an aliquot was taken for protein (Bradford assay). The column was eluted with a 60-min gradient of 27–30% acetonitrile in 0.1% trifluoroacetic acid at 1 ml/min. Fractions (1 min) were collected and aliquots were dried with a Speed Vac concentration before CCK RIA (47). Sulfated CCK 12 and 22 were synthesized as described elsewhere (48).

[3H]Thymidine Incorporation Assays
For the last 18 h, [3H]thymidine was added to the culture media (1 µCi/ml). On d 3 post treatment, islets were washed three times with ice-cold PBS, and DNA and protein were precipitated by addition of 10% trichloroacetic acid. The precipitate was solubilized in 0.3 N NaOH, and the radioactivity was measured using a liquid scintillation counter. A fraction of the solubilized product was kept to measure total protein by the Bradford assay (49). Sample counts were individually normalized to protein, and an average for each treatment group was determined.

Glucose-Stimulated Insulin Secretion
On d 3 after treatment, mouse islets were washed in mouse Krebs Ringer bicarbonate (KRB) secretion buffer containing 0.5% BSA, 118.41 mM NaCl, 4.69 mM KCl, 1.18 mM MgSO4, 1.18 mM KH2PO4, 25 mM NaHCO3, 5 mM HEPES, and 2.52 mM CaCl2 with 1.67 mM glucose for 45 min. Islets were then either incubated in KRB secretion buffer with 1.67 mM glucose or 16.7 mM glucose for 45 min. Insulin concentrations were measured by ELISA and normalized to total insulin content.

On posttreatment d 3, human islets were washed for 1 h in human KRB secretion buffer containing 4.38 mM KCl, 1.2 mM MgSO4, 1.5 mM KH2PO4, 129 mM NaCl, 5 mM NaHCO3, 10 mM HEPES, 3.11 mM CaCl2, and 0.25% BSA with 2.8 mM glucose. Islets were then incubated for 1 h in human KRB secretion buffer containing 2.8 mM glucose or 16.7 mM glucose. Insulin concentrations were measured by RIA and normalized to total islet protein.

Immunofluorescence Staining
Human and mouse islets were treated identically to [3H]thymidine incorporation experiments, but were incubated in 10 mM 5-bromo-2'-deoxyuridine (BrdU) for the final 18 h of the experiment. Islets were washed three times with 1 ml of PBS. Islets were fixed in Bouin’s solution for 2 h and maintained in 10% neutral-buffered formalin. After formalin removal, 50 ml of Affi-Gel blue bead slurry (Bio-Rad Laboratories, Hercules, CA) was added to the islets to aid in visualization during sectioning. The islet and bead slurry was embedded in paraffin. Serial sections (5 µm) on glass slides were deparaffinized with xylene and rehydrated in a graded series of ethanol. For mouse islets, BrdU (Calbiochem, La Jolla, CA; NA20, 1:50) and insulin (Sigma Chemical Co., St Louis, MO; I8510, 1:500) were immunostained. For human islets, slides were boiled for 13 min in Vector H-3300 antigen retrieval solution (Vector Laboratories, Inc., Burlingame, CA) after rehydration. BrdU (Invitrogen, Carlsbad, CA; A21301MP, 1:100), glucagon (Santa Cruz Biotechnology, Inc., Santa Cruz, CA; sc-13091, 1:50), and insulin were immunostained. Islet sections were photographed on x60 confocal microscopy. Pictures were blinded and scored for BrdU-positive nuclei surrounded by insulin- or glucagons-positive staining.

Flow Cytometry Analysis
Islets were collected on posttreatment d 3. Islets were washed in modified KRB buffer (137 mM NaCl, 4.7 mM KCl, 1.2 mM KH2PO4, 1.2 mM MgSO4, 2.5 mM CaCl2, 25 mM NaHCO3, 10 mM HEPES, 3.3 mM glucose). Islets were dispersed into single cells by incubation in Cell Dissociation Solution (Sigma) at 37 C with shaking. Cells were fixed in 100% ethanol and stored at –20 C. Cells were immunostained for insulin (Sigma; I8510, 1:100) in modified KRB with 0.1% BSA and 0.1% Tween 20, followed by treatment with ribonuclease A (10 µg/ml) at 37 C for 30 min. Cells were then stained overnight with propidium iodide at 50 µg/ml at 4 C. Insulin-positive cells and cell cycle stage were identified on a FACSCalibur flow cytometer (Becton Dickinson, Franklin Lakes, NJ). Flow cytometry data was analyzed with CellQuestPro (Becton Dickinson), FlowJo (Tree Star, Inc.), and ModFit (Verity Software, Topsham, ME) software to determine the percentage of cells in each phase of the cell cycle.

Quantitative RT-PCR
Islets were washed in PBS and then homogenized in RLT buffer (QIAGEN, Chatsworth, CA). RNA was isolated with the RNeasy kit (QIAGEN) and cDNA prepared with the Superscript III kit (Invitrogen). mRNA measurements were performed by SYBR quantitative RT-PCR, and all values were normalized to β-actin. Primer sequences can be found in supplemental Tables 1 and 2 published as supplemental data on The Endocrine Society’s Journals Online web site at http://mend. endojournals.org. Fold changes were determined for each independent mouse or human and averaged.

Western Blotting
Islets were washed in PBS and lysed in 20 mM Tris HCl, 10 mM EDTA, and 1% Triton X-100 containing 10 µg/ml leupeptin, 5 µg/ml aprotinin, 5 µg/ml pepstatin A, 100 µM pefablock, and 1 mM sodium orthovanadate. Whole islet lysate (20–50 µg) was separated on 4–15% Tris HCl gradient gels (Bio-Rad) and transferred to polyvinylidine difluoride membranes. Membranes were blocked in 5% milk Tris-buffered saline with 0.25% Tween 20. Primary antibodies and dilutions can be found in supplemental Table 2. Blots were quantitated by densitometry with the QuantTL program. Fold changes were determined for each individual mouse or human and averaged.

Statistical Methods
Significant changes for [3H]thymidine- and glucose-stimulated insulin secretions between untreated, AdCMV-β-Gal, and AdCMV-CCK treated groups were determined by one-way ANOVA followed by a Bonferroni-corrected t test. BrdU, flow cytometry, quantitative RT-PCR, and Western blot differences between AdCMV-β-Gal- and AdCMV-CCK-treated groups were determined by Student’s paired t test.


   ACKNOWLEDGMENTS
 
We thank Kathy Schell and the rest of the staff at the University of Wisconsin Comprehensive Cancer Center flow cytometry facility for assistance with protocol development and data collection. For human islets and resources, we specifically thank Dr. Matt Hanson and Dr. Luis Fernandez and the Islet Cell Resource (ICR) centers Basic Science Islet Distribution Program, including the following ICR centers: University of Wisconsin-Madison, University of Alabama at Birmingham, University of Illinois at Chicago, University of Pennsylvania, University of Minnesota, and Southern California Islet Consortium. We also thank Klaus Kaestner for cyclin and cdk antibodies and Kouki Kitagawa at Niigata University of Pharmacy and Applied Life Sciences (Japan) for synthesis of sulfated CCK12 and CCK12 amides.


   FOOTNOTES
 
This work was supported by Juvenile Diabetes Research Foundation Grant 17-2007-1026 (to A.D.A. and C.B.N.). A.D.A. was additionally supported by National Institutes of Health Grants DK 66539 and DK 58037. J.A.L. was supported by a National Human Genome Research Institute training grant to the Genomic Sciences Training Program (5T32HG002760). P.W.R. and D.B.D. were supported by National Institute on Aging training grant T32 AG20013.

Disclosure Statement: The authors have nothing to disclose.

First Published Online October 9, 2008

1 J.A.L. and P.W.R. contributed equally to this work. Back

Abbreviations: BrdU, 5-Bromo-2'-deoxyuridine; CCK, cholecystokinin; CMV, cytomegalovirus; GLP-1, glucagon-like peptide 1; HGF, hepatocyte growth factor; KRB, Krebs Ringer bicarbonate; PI3K; phosphoinositol 3-kinase; PKA, protein kinase A; STAT, signal transducer and activator of transcription; TFF3, trefoil factor 3.

Received for publication July 24, 2008. Accepted for publication October 1, 2008.


   REFERENCES
TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 

  1. Ryan EA, Lakey JR, Paty BW, Imes S, Korbutt GS, Kneteman NM, Bigam D, Rajotte RV, Shapiro AM 2002 Successful islet transplantation: continued insulin reserve provides long-term glycemic control. Diabetes 51:2148–2157[Abstract/Free Full Text]
  2. Ryan EA, Lakey JR, Rajotte RV, Korbutt GS, Kin T, Imes S, Rabinovitch A, Elliott JF, Bigam D, Kneteman NM, Warnock GL, Larsen I, Shapiro AM 2001 Clinical outcomes and insulin secretion after islet transplantation with the Edmonton protocol. Diabetes 50:710–719[Abstract/Free Full Text]
  3. Shapiro AM, Lakey JR, Ryan EA, Korbutt GS, Toth E, Warnock GL, Kneteman NM, Rajotte RV 2000 Islet transplantation in seven patients with type 1 diabetes mellitus using a glucocorticoid-free immunosuppressive regimen. N Engl J Med 343:230–238[Abstract/Free Full Text]
  4. Garcia-Ocana A, Takane KK, Syed MA, Philbrick WM, Vasavada RC, Stewart AF 2000 Hepatocyte growth factor overexpression in the islet of transgenic mice increases β cell proliferation, enhances islet mass, and induces mild hypoglycemia. J Biol Chem 275:1226–1232[Abstract/Free Full Text]
  5. Beattie GM, Otonkoski T, Lopez AD, Hayek A 1997 Functional β-cell mass after transplantation of human fetal pancreatic cells: differentiation or proliferation? Diabetes 46:244–248[Abstract]
  6. Beattie GM, Rubin JS, Mally MI, Otonkoski T, Hayek A 1996 Regulation of proliferation and differentiation of human fetal pancreatic islet cells by extracellular matrix, hepatocyte growth factor, and cell-cell contact. Diabetes 45:1223–1228[Abstract]
  7. Buteau J, Roduit R, Susini S, Prentki M 1999 Glucagon-like peptide-1 promotes DNA synthesis, activates phosphatidylinositol 3-kinase and increases transcription factor pancreatic and duodenal homeobox gene 1 (PDX-1) DNA binding activity in β (INS-1)-cells. Diabetologia 42:856–864[CrossRef][Medline]
  8. Xu G, Stoffers DA, Habener JF, Bonner-Weir S 1999 Exendin-4 stimulates both β-cell replication and neogenesis, resulting in increased β-cell mass and improved glucose tolerance in diabetic rats. Diabetes 48:2270–2276[Abstract]
  9. Friedrichsen BN, Neubauer N, Lee YC, Gram VK, Blume N, Petersen JS, Nielsen JH, Moldrup A 2006 Stimulation of pancreatic β-cell replication by incretins involves transcriptional induction of cyclin D1 via multiple signalling pathways. J Endocrinol 188:481–492[Abstract/Free Full Text]
  10. Vasavada RC, Garcia-Ocana A, Zawalich WS, Sorenson RL, Dann P, Syed M, Ogren L, Talamantes F, Stewart AF 2000 Targeted expression of placental lactogen in the β cells of transgenic mice results in β cell proliferation, islet mass augmentation, and hypoglycemia. J Biol Chem 275:15399–15406[Abstract/Free Full Text]
  11. Vasavada RC, Wang L, Fujinaka Y, Takane KK, Rosa TC, Mellado-Gil JM, Friedman PA, Garcia-Ocana A 2007 Protein kinase C-{zeta} activation markedly enhances β-cell proliferation: an essential role in growth factor mediated β-cell mitogenesis. Diabetes 56:2732–2743[Abstract/Free Full Text]
  12. Fueger PT, Schisler JC, Lu D, Babu DA, Mirmira RG, Newgard CB, Hohmeier HE 2008 Trefoil factor 3 stimulates human and rodent pancreatic islet β-cell replication with retention of function. Mol Endocrinol 22:1251–1259[Abstract/Free Full Text]
  13. Kuntz E, Pinget M, Damge P 2004 Cholecystokinin octapeptide: a potential growth factor for pancreatic β cells in diabetic rats. JOP 5:464–475[Medline]
  14. Chen S, Turner S, Tsang E, Stark J, Turner H, Mahsut A, Keifer K, Goldfinger M, Hellerstein MK 2007 Measurement of pancreatic islet cell proliferation by heavy water labeling. Am J Physiol Endocrinol Metab 293:E1459–E1464
  15. Lan H, Rabaglia ME, Stoehr JP, Nadler ST, Schueler KL, Zou F, Yandell BS, Attie AD 2003 Gene expression profiles of nondiabetic and diabetic obese mice suggest a role of hepatic lipogenic capacity in diabetes susceptibility. Diabetes 52:688–700[Abstract/Free Full Text]
  16. Schisler JC, Fueger PT, Babu DA, Hohmeier HE, Tessem JS, Lu D, Becker TC, Naziruddin B, Levy M, Mirmira RG, Newgard CB 2008 Stimulation of human and rat islet β-cell proliferation with retention of function by the homeodomain transcription factor Nkx6.1. Mol Cell Biol 28:3465–3476[Abstract/Free Full Text]
  17. Hohmeier HE, Newgard CB 2005 Islets for all? Nat Biotechnol 23:1231–1232[CrossRef][Medline]
  18. Fatrai S, Elghazi L, Balcazar N, Cras-Meneur C, Krits I, Kiyokawa H, Bernal-Mizrachi E 2006 Akt induces β-cell proliferation by regulating cyclin D1, cyclin D2, and p21 levels and cyclin-dependent kinase-4 activity. Diabetes 55:318–325[Abstract/Free Full Text]
  19. Fung TK, Siu WY, Yam CH, Lau A, Poon RY 2002 Cyclin F is degraded during G2-M by mechanisms fundamentally different from other cyclins. J Biol Chem 277:35140–35149[Abstract/Free Full Text]
  20. Cozar-Castellano I, Weinstock M, Haught M, Velazquez-Garcia S, Sipula D, Stewart AF 2006 Evaluation of β-cell replication in mice transgenic for hepatocyte growth factor and placental lactogen: comprehensive characterization of the G1/S regulatory proteins reveals unique involvement of p21cip. Diabetes 55:70–77[Abstract/Free Full Text]
  21. Kopin AS, Mathes WF, McBride EW, Nguyen M, Al-Haider W, Schmitz F, Bonner-Weir S, Kanarek R, Beinborn M 1999 The cholecystokinin-A receptor mediates inhibition of food intake yet is not essential for the maintenance of body weight. J Clin Invest 103:383–391[Medline]
  22. Langhans N, Rindi G, Chiu M, Rehfeld JF, Ardman B, Beinborn M, Kopin AS 1997 Abnormal gastric histology and decreased acid production in cholecystokinin-B/gastrin receptor-deficient mice. Gastroenterology 112:280–286[CrossRef][Medline]
  23. Weir GC, Bonner-Weir S 2004 Five stages of evolving β-cell dysfunction during progression to diabetes. Diabetes 53(Suppl 3):S16–S21
  24. Wajchenberg BL 2007 β-Cell failure in diabetes and preservation by clinical treatment. Endocr Rev 28:187–218[Abstract/Free Full Text]
  25. Prentki M, Nolan CJ 2006 Islet β cell failure in type 2 diabetes. J Clin Invest 116:1802–1812[CrossRef][Medline]
  26. Rao P, Roccisana J, Takane KK, Bottino R, Zhao A, Trucco M, Garcia-Ocana A 2005 Gene transfer of constitutively active Akt markedly improves human islet transplant outcomes in diabetic severe combined immunodeficient mice. Diabetes 54:1664–1675[Abstract/Free Full Text]
  27. Path G, Opel A, Gehlen M, Rothhammer V, Niu X, Limbert C, Romfeld L, Hugl S, Knoll A, Brendel MD, Bretzel RG, Seufert J 2006 Glucose-dependent expansion of pancreatic β-cells by the protein p8 in vitro and in vivo. Am J Physiol Endocrinol Metab 291:E1168–E1176
  28. Brun T, Hu He KH, Lupi R, Boehm B, Wojtusciszyn A, Sauter N, Donath M, Marchetti P, Maedler K, Gauthier BR 2007 The diabetes-linked transcription factor Pax4 is expressed in human pancreatic islets and is activated by mitogens and GLP-1. Hum Mol Genet 17:478–489[CrossRef][Medline]
  29. Ryan EA, Paty BW, Senior PA, Bigam D, Alfadhli E, Kneteman NM, Lakey JR, Shapiro AM 2005 Five-year follow-up after clinical islet transplantation. Diabetes 54:2060–2069[Abstract/Free Full Text]
  30. Ahren B, Hedner P, Lundquist I 1983 Interaction of gastric inhibitory polypeptide (GIP) and cholecystokinin (CCK-8) with basal and stimulated insulin secretion in mice. Acta Endocrinol (Copenh) 102:96–102[Abstract/Free Full Text]
  31. Fehmann HC, Goke R, Goke B, Bachle R, Wagner B, Arnold R 1991 Priming effect of glucagon-like peptide-1 (7–36) amide, glucose-dependent insulinotropic polypeptide and cholecystokinin-8 at the isolated perfused rat pancreas. Biochim Biophys Acta 1091:356–363[Medline]
  32. Hildebrand P, Ensinck JW, Ketterer S, Delco F, Mossi S, Bangerter U, Beglinger C 1991 Effect of a cholecystokinin antagonist on meal-stimulated insulin and pancreatic polypeptide release in humans. J Clin Endocrinol Metab 72:1123–1129[Abstract/Free Full Text]
  33. Rane SG, Dubus P, Mettus RV, Galbreath EJ, Boden G, Reddy EP, Barbacid M 1999 Loss of Cdk4 expression causes insulin-deficient diabetes and Cdk4 activation results in β-islet cell hyperplasia. Nat Genet 22:44–52[CrossRef][Medline]
  34. Kushner JA, Ciemerych MA, Sicinska E, Wartschow LM, Teta M, Long SY, Sicinski P, White MF 2005 Cyclins D2 and D1 are essential for postnatal pancreatic β-cell growth. Mol Cell Biol 25:3752–3762[Abstract/Free Full Text]
  35. Georgia S, Bhushan A 2004 β-Cell replication is the primary mechanism for maintaining postnatal β cell mass. J Clin Invest 114:963–968[CrossRef][Medline]
  36. Zhang X, Gaspard JP, Mizukami Y, Li J, Graeme-Cook F, Chung DC 2005 Overexpression of cyclin D1 in pancreatic β-cells in vivo results in islet hyperplasia without hypoglycemia. Diabetes 54:712–719[Abstract/Free Full Text]
  37. Cozar-Castellano I, Takane KK, Bottino R, Balamurugan AN, Stewart AF 2004 Induction of β-cell proliferation and retinoblastoma protein phosphorylation in rat and human islets using adenovirus-mediated transfer of cyclin- dependent kinase-4 and cyclin D1. Diabetes 53:149–159[Abstract/Free Full Text]
  38. Friedrichsen BN, Richter HE, Hansen JA, Rhodes CJ, Nielsen JH, Billestrup N, Moldrup A 2003 Signal transducer and activator of transcription 5 activation is sufficient to drive transcriptional induction of cyclin D2 gene and proliferation of rat pancreatic β-cells. Mol Endocrinol 17:945–958[Abstract/Free Full Text]
  39. Friedrichsen BN, Galsgaard ED, Nielsen JH, Moldrup A 2001 Growth hormone- and prolactin-induced proliferation of insulinoma cells, INS-1, depends on activation of STAT5 (signal transducer and activator of transcription 5). Mol Endocrinol 15:136–148[Abstract/Free Full Text]
  40. Rulifson IC, Karnik SK, Heiser PW, ten Berge D, Chen H, Gu X, Taketo MM, Nusse R, Hebrok M, Kim SK 2007 Wnt signaling regulates pancreatic β cell proliferation. Proc Natl Acad Sci USA 104:6247–6252[Abstract/Free Full Text]
  41. Keller MP, Choi Y, Wang P, Davis DB, Rabaglia ME, Oler AT, Stapleton DS, Argmann C, Schueler KL, Edwards S, Steinberg HA, Neto EC, Kleinhanz R, Turner S, Hellerstein MK, Schadt EE, Yandell BS, Kendziorski C, Attie AD 2008 A gene expression network model of type 2 diabetes links cell cycle regulation in islets with diabetes susceptibility. Genome Res 18:706–716[Abstract/Free Full Text]
  42. Dufresne M, Seva C, Fourmy D 2006 Cholecystokinin and gastrin receptors. Physiol Rev 86:805–847[Abstract/Free Full Text]
  43. Julien S, Laine J, Morisset J 2004 The rat pancreatic islets: a reliable tool to study islet responses to cholecystokinin receptor occupation. Regul Pept 121:73–81[CrossRef][Medline]
  44. Becker TC, Noel RJ, Coats WS, Gomez-Foix AM, Alam T, Gerard RD, Newgard CB 1994 Use of recombinant adenovirus for metabolic engineering of mammalian cells. Methods Cell Biol 43(Pt A):161–189
  45. Lacourse KA, Swanberg LJ, Gillespie PJ, Rehfeld JF, Saunders TL, Samuelson LC 1999 Pancreatic function in CCK-deficient mice: adaptation to dietary protein does not require CCK. Am J Physiol 276:G1302–G1309
  46. Rabaglia ME, Gray-Keller MP, Frey BL, Shortreed MR, Smith LM, Attie AD 2005 {alpha}-Ketoisocaproate-induced hypersecretion of insulin by islets from diabetes-susceptible mice. Am J Physiol Endocrinol Metab 289:E218–E224
  47. Beinfeld MC, Meyer DK, Eskay RL, Jensen RT, Brownstein MJ 1981 The distribution of cholecystokinin immunoreactivity in the central nervous system of the rat as determined by radioimmunoassay. Brain Res 212:51–57[CrossRef][Medline]
  48. Kitagawa K, Aida C, Fujiwara H, Yagami T, Futaki S, Kogire M, Ida J, Inoue K 2001 Facile solid-phase synthesis of sulfated tyrosine-containing peptides: total synthesis of human big gastrin-II and cholecystokinin (CCK)-39. J Org Chem 66:1–10[CrossRef][Medline]
  49. Bradford MM 1976 A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding. Anal Biochem 72:248–254[CrossRef][Medline]



This article has been cited by other articles:


Home page
 DiabetesHome page
S. Georgia, C. Hinault, D. Kawamori, J. Hu, J. Meyer, M. Kanji, A. Bhushan, and R. N. Kulkarni
Cyclin D2 Is Essential for the Compensatory {beta}-Cell Hyperplastic Response to Insulin Resistance in Rodents
Diabetes, April 1, 2010; 59(4): 987 - 996.
[Abstract] [Full Text] [PDF]

Home page
 Mol. Endocrinol.Home page
J. A. Lavine, P. W. Raess, D. B. Davis, M. E. Rabaglia, B. K. Presley, M. P. Keller, M. C. Beinfeld, A. S. Kopin, C. B. Newgard, and A. D. Attie
Contamination with E1A-Positive Wild-Type Adenovirus Accounts for Species-Specific Stimulation of Islet Cell Proliferation by CCK: A Cautionary Note
Mol. Endocrinol., February 1, 2010; 24(2): 464 - 467.
[Abstract] [Full Text] [PDF]

This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow Supplemental Data
Right arrow A retraction has been published
Right arrow Submit a related Letter to the Editor
Right arrow Alert me when this article is cited
Right arrow Alert me when eLetters are posted
Right arrow Alert me if a correction is posted
Right arrow Citation Map
Services
Right arrow Email this article to a friend
Right arrow Similar articles in this journal
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Right arrow Reprints, Permissions and Rights
Citing Articles
Right arrow Citing Articles via HighWire
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Lavine, J. A.
Right arrow Articles by Attie, A. D.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Lavine, J. A.
Right arrow Articles by Attie, A. D.

HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS
Endocrinology Endocrine Reviews J. Clin. End. & Metab.
Molecular Endocrinology Recent Prog. Horm. Res. All Endocrine Journals