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Endocrinology, doi:10.1210/en.2007-0387
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Endocrinology Vol. 148, No. 11 5520-5530
Copyright © 2007 by The Endocrine Society

c-Kit in Early Onset of Diabetes: A Morphological and Functional Analysis of Pancreatic β-Cells in c-KitW-v Mutant Mice

Mansa Krishnamurthy1, Farzam Ayazi1, Jinming Li1, Alexander W. Lyttle, Michael Woods, Yuexiu Wu, Siu-Pok Yee and Rennian Wang

Children’s Health Research Institute (M.K., F.A., J.L., A.W.L., M.W., Y.W., R.W.), Departments of Pathology (M.K.), Physiology and Pharmacology (F.A., J.L., Y.W., R.W.), Medicine (R.W.), Biochemistry (S.-P.Y.), and Oncology (S.-P.Y.), University of Western Ontario, London, Ontario, Canada N6C 2V5

Address all correspondence and requests for reprints to: Dr. Rennian Wang, Victoria Laboratory Centre, Room A5-140, 800 Commissioners Road East, London, Ontario, Canada N6C 2V5. E-mail: rwang{at}uwo.ca.


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
c-Kit tyrosine receptor kinase, a well-established stem cell marker, is expressed in a variety of tissues including the pancreas. The involvement of c-Kit in fetal rat and human endocrine pancreatic development, survival, and function has been well characterized but primarily using in vitro experimental approaches. Therefore, the aim of the current study was to examine whether deficiency of a functional c-Kit receptor would have physiological and functional implications in vivo. We characterized the c-Kit mutant mouse, c-KitW-v/+, to evaluate the in vivo role of c-Kit in β-cell growth and function. Here we report that male c-KitW-v/+ mice, at 8 wk of age, showed high fasting blood glucose levels and impaired glucose tolerance, which was associated with low levels of insulin secretion after glucose stimulation in vivo and in isolated islets. Morphometric analysis revealed that β-cell mass was significantly reduced (50%) in male c-KitW-v/+ mice when compared with controls (c-Kit+/+) (P < 0.05). In parallel, a reduction in pancreatic duodenal homeobox-1 and insulin gene expression in whole pancreas as well as isolated islets of c-KitW-v/+ male mice was noted along with a decrease in pancreatic insulin content. Furthermore, the reduction in β-cell mass in male c-KitW-v/+ mice was associated with a decrease in β-cell proliferation. Interestingly, these changes were not observed in female c-KitW-v/+ mice until 40 wk of age. Our results clearly demonstrate that the c-Kit receptor is involved in the regulation of glucose metabolism, likely through an important role in β-cell development and function.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
THE C-KIT TYROSINE receptor kinase, a well-established stem cell marker, and its ligand, stem cell factor (SCF), are expressed in a variety of body tissues. Binding of SCF induces homodimerization of the c-Kit receptor (1, 2, 3, 4), resulting in an increase in receptor autophosphorylation activity and recruitment of the c-Kit signaling complex (5). These events, which lead to the activation of a number of intracellular pathways, have been implicated in the survival, proliferation, and differentiation of stem cells in hematopoiesis, gametogenesis, and melanogenesis (6, 7).

In the pancreas, c-Kit is involved in the development, survival, and function of rodent islets of Langerhans and, more importantly, is essential for β-cell survival (2, 8). In vitro studies have shown that fetal rat β-cells produced increased levels of insulin upon stimulation with exogenous SCF (2). In addition, our own studies revealed pronounced changes in the distribution and cell population dynamics of c-Kit-expressing cells in both ductal and islet clusters in the developing rat and human pancreas (9, 10). We have also shown that c-Kit-expressing cells can be rapidly expanded in vitro from isolated islets of neonatal rat pancreas and are capable of reforming islet-like clusters that secrete insulin (11). These results, taken together, indicate that c-Kit-expressing cells can act as islet precursors. In addition, c-Kit has been shown to play a role in islet cell neogenesis: the expression of c-Kit after rat pancreatic duct ligation was only found in ductal cells of the ligated portion during islet cell neogenesis (12). The increase in c-Kit+ cells was observed before new islets budding off from the ductal epithelium in association with an up-regulation in pancreatic duodenal homeobox (Pdx)-1, pre-B-cell leukemia homeobox-1 (Pbx-1), myeloid ecotropic viral integration site 1 homolog 2 (Meis2), and Nk 2 homeobox 2 (Nkx2.2) expression. Furthermore, we have demonstrated that addition of exogenous SCF to human fetal islet-epithelial clusters promoted β-cell development by increasing SCF/c-Kit interactions and, in turn, augmenting Pdx-1 gene and protein expression (13).

Although the role of c-Kit has been characterized in developing rat and human pancreas (3, 9, 10, 14), mutated forms of the c-Kit receptor are not available in these model systems, which would allow for in vivo assessment of c-Kit’s physiological and functional roles. In the mouse, the c-Kit point mutation, W-v, which leads to a substitution of threonine by a methionine at position 660, alters the ATP-binding domain of the c-Kit receptor tyrosine kinase. This greatly diminishes the receptor’s kinase activity without altering its binding site. As expected, c-KitW-v mutant mice exhibit distinct defects in hematopoiesis, melanogenesis, and gametogenesis. Homozygous c-KitW-v (c-KitW-v/W-v) mice completely lack fur pigmentation (Fig. 1AGo) and are severely anemic and infertile because they possess much smaller gonads with no gametes. Given the essential role of c-Kit in islet development, survival, and function, we anticipated that characterization of the c-KitW-v mutant mice would provide a much better understanding of the in vivo functional role of c-Kit in glucose metabolism and islet cell development, survival, and function. In the present study, we characterized heterozygous c-KitW-v (c-KitW-v/+) mice and investigated whether an impaired c-Kit receptor would affect islet and β-cell mass and, in turn, glucose metabolism. We now report that c-KitW-v/+ males possess a reduced β-cell mass at 4 wk of age. In addition, these mice consistently exhibit high fasting blood glucose levels, are glucose intolerant, and demonstrate reduced levels of Pdx-1 and insulin gene expression. The majority of these changes were not observed in the c-KitW-v/+ females until 40 wk of age, suggesting that a compensatory mechanism, likely involving differential sex hormone expression, exists whereby β-cell mass in females is rescued from the consequences of the c-KitW-v mutation. A better understanding of the role of c-Kit in islet cell development and survival as well as local and systemic effects of glucose metabolism may enable the development of new strategies for diabetes treatment.


Figure 1
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  		FIG. 1. Genotype and phenotype of c-Kit+/+, c-KitW-v/+, and c-KitW-v/W-v mice. A, Fur pigmentation of 8-wk-old c-Kit+/+, c-KitW-v/+, and c-KitW-v/W-v mice and c-Kit expression in the pancreas in all three genotypes as determined by Western blot (top) and qRT-PCR (bottom). IB, Immunoblot. Body weight (B) and fasting blood glucose levels (C) of male and female c-Kit+/+ and c-KitW-v/+ mice at different time points in the study are shown. **, P < 0.01 vs. female mice in B; **, P < 0.01 vs. male c-Kit+/+ and female mice in C (n = 5–15).

 

   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Heterozygotic C57BL/6J/KitW-v mice
Heterozygous C57BL/6J/KitW-v mice (4–6 wk old) were obtained from the Jackson Laboratory (Bar Harbor, ME; stock no. 000049) and bred to obtain homozygous wild-type (c-Kit+/+), heterozygous mutant (c-KitW-v/+),and homozygous mutant (c-KitW-v/W-v) mice. Mice were genotyped by their distinct differences in fur pigmentation: black for c-Kit+/+, piebaldism for c-KitW-v/+, and white for c-KitW-v/W-v mice (Fig. 1AGo). The pancreatic expression of c-Kit at both the mRNA and protein level was similar among the three genotypes (Fig. 1AGo). Mice were maintained in an environment of constant temperature and regular 12-h day, 12-h night cycles. Body weight and fasting blood glucose levels were monitored on a regular basis. Mice were killed at 4, 8, and 16 wk after birth. Pancreata were carefully dissected, weighed, and processed for either histological studies or RNA and protein analyses. A minimum of five and a maximum of 19 animals were used per age per group for each experiment conducted. All protocols were approved by the Animal User Subcommittee at the University of Western Ontario in accordance with the guidelines of the Canadian Council of Animal Care.

Intraperitoneal glucose tolerance test (IPGTT), ip insulin tolerance test (IPITT), and glucose-stimulated insulin secretion
IPGTTs were performed using mice at 4, 8, 16, and 40 wk of age, as described previously (15, 16, 17). Briefly, after 4 h of fasting, an ip injection of glucose [D-(+)-glucose; dextrose; Sigma, St. Louis, MO] of 2 mg/g of body weight was administrated to each mouse. Blood glucose levels were examined at 0, 15, 30, 60, 90, and 120 min after the injection. To quantify glucose responsiveness, area under the curve (AUC) was calculated using the trapezoidal method (18). For the IPITT, 8-wk-old mice from both c-Kit+/+ and c-KitW-v/+ groups were fasted for 4 h, followed by an injection of normal human insulin (Humalin 1 U/kg of body weight; Eli Lilly, Toronto, Ontario, Canada), and blood glucose levels were measured at 0, 15, 30, and 60 min.

To examine glucose-stimulated insulin secretion in 8-wk-old male c-Kit+/+ and c-KitW-v/+ mice, blood was collected from the tail vein after 4 h of fasting (0 min), and at 5 and 35 min after glucose stimulation, using heparinized capillary tubes (The Microvette CB 300 capillary tube; Sarstedt Inc., Montréal, Québec, Canada). Plasma samples were immediately obtained by centrifuging at 3000 rpm for 10 min and stored at –20 C. To assess whether the c-Kit mutation had an effect on the islet of Langerhans, glucose stimulated insulin secretion on isolated islets from 8-wk-old male c-Kit+/+ and c-KitW-v/+ mice was performed. Hand-picked islets isolated after an intraductal collagenase V injection, as previously described (11), were cultured in RPMI 1640 medium supplemental with 10% fetal bovine serum (Invitrogen, Burlington, Ontario, Canada) for an hour and subjected to an acute glucose challenge (11): islets were incubated in RPMI 1640 medium (Sigma) containing 2.2 mM glucose for 2 h and then incubated with 22 mM glucose for 1 h. Insulin secretion levels at both time points were assessed by ELISA. A static glucose stimulation index was calculated by dividing the insulin output from the high glucose incubation by the insulin output during low-glucose (2.2 mM) incubation (11).

Immunohistochemistry and double immunofluorescence
Pancreata collected from both c-Kit+/+ and c-KitW-v/+ mice at 4 and 8 wk of age were fixed in 4% paraformaldehyde followed by a standard protocol of dehydration and paraffin embedding. Pancreatic tissue sections (4 µm thick) were prepared from the entire length of the pancreas to monitor regional changes in islet distribution and islet-cell composition (19). Two sets of six serial sections at 50-µm intervals were stained with hematoxylin-eosin or incubated with mouse monoclonal antibodies against human insulin or glucagon (Sigma) using the AB complex method (Histostain plus kit; Zymed, San Francisco, CA), as described previously (19). Antibody staining was visualized using diaminobenzidine (Zymed). Controls were performed by omitting primary or secondary antibody.

Double immunofluorescence of pancreatic sections was performed using antibodies against Ki67 (Abcam, Cambridge, MA) and insulin to examine the proliferative capacity of β-cells as well as insulin and Pdx-1 (provided by Dr. Christopher V. Wright, University of Vanderbilt, Nashville, TN) for coexpression analysis in both c-Kit+/+ and c-KitW-v/+ mice. The secondary antibodies, fluorescein (fluorescein isothiocyanate) (antimouse) and rhodamine (tetramethylrhodamine isothiocyanate) (antirabbit), were obtained from Jackson ImmunoResearch Laboratories (West Grove, PA). Double-labeled images were recorded by a Leica DMIRE2 fluorescence microscope with Openlab image software (Improvision, Lexington, MA).

Morphometric analysis
Quantitative evaluation was performed using computer-assisted image analysis (10). Areas of {alpha}-cells, β-cells, and pancreatic islets in sections from both c-Kit+/+ and c-KitW-v/+ mice at 4 and 8 wk of age were traced manually in cases in which two pancreatic sections per animal were examined with a minimum of five pancreata per age group per experimental group. Islets of Langerhans were identified with hematoxylin-eosin staining, whereas {alpha}- and β-cells were defined via immunohistochemical staining. Total {alpha}- and β-cell mass was determined using previously described methods (19): Aβ/AP = Mβ/MP, where Aβ and Mβ are total insulin+ area and β-cell mass, and AP and MP are total pancreatic section area and pancreatic mass, with the same logic for measuring {alpha}-cell mass. The percentage of Ki67-labeling index of β-cells was determined by counting the total number of insulin-positive cells and the total Ki67-insulin double-positive cells from at least 10 islets randomly imaged from each pancreatic section. A minimum of five pancreata per age group per experimental group was analyzed. The data were presented as a percentage of the total number of insulin-positive cells counted.

Measurement of insulin secretion and pancreatic insulin content
Pancreatic insulin was extracted in an ethanol-acid solution (165 mM HCl in 75% ethanol). Plasma and pancreatic insulin content as well as insulin content of isolated islets were measured using a mouse ultrasensitive insulin ELISA kit (ALPCO, Salem, NH) with a sensitivity of 0.15 ng/ml, according to the manufacturer’s instructions. DNA content of the tissue samples was determined along with the insulin content using a Multiskan Spectrum microplate spectrophotometer (Thermo Electron Corp., Waltham, MA). Insulin content was expressed as nanograms per microgram DNA (11).

Western blot analysis
Protein expression of c-Kit and Pdx-1 from male and female c-Kit+/+ and c-KitW-v/+ mice at 8 wk of age was analyzed by homogenization of pancreata in a Nonidet-P40 lysis buffer, as described previously (20). Equal amounts (50 µg) of lysate protein from each experimental group were separated by 5% (for c-Kit) or 12% (for Pdx-1) SDS-PAGE and then transferred onto a nitrocellulose membrane (Bio-Rad Laboratories, Mississauga, Ontario, Canada). Membranes were then incubated with primary antibodies: rabbit anti-c-Kit (C-19; Santa Cruz Biotechnology, Santa Cruz, CA) and Pdx-1, followed by incubation with a secondary antibody of goat antirabbit IgG conjugated to horseradish peroxidase (Santa Cruz Biotechnology). As a control for sample loading, the blot was also probed with antibodies against mouse calnexin (BD Biosciences, Mississauga, Ontario, Canada) and β-actin (Sigma). Membranes were incubated in chemiluminescence reagents (PerkinElmer, Wellesley, MA) and exposed to BioMax MR Film (Kodak, Rochester, NY) to reveal protein bands. Densitometric quantification of bands at subsaturating levels was performed using the Syngenetool gel analysis software (Syngene, Cambridge, UK) and normalized to the loading control of β-actin. Data were expressed as the relative protein expression normalized to loading control (20).

RNA extraction, real-time RT-PCR (qRT-PCR)
Total RNA was extracted from whole pancreata of both c-Kit+/+ and c-KitW-v/+ mice at 8 wk of age using TRIZOL reagent (Invitrogen), as described previously (10). RNA from isolated islets was extracted using the RNAqueous-4PCR kit (Ambion, Austin, TX), according to the manufacturer’s instructions (11). For each reverse transcription reaction 2 µg of total RNA from whole pancreatic tissue and isolated islets were used with oligo(dT) primers and Superscript reverse transcriptase (Invitrogen). Sequences of PCR primers used for RT-PCR with expected size of product, are as follows: c-Kit, forward, 5'-TTA CAT AGA CCC GAC GCA ACT T-3', reverse, 5'-TTT GAG CAT CTT CAC GGC AAC T-3' (172 bp); Pdx-1, forward, 5'-CCA CCC CAG TTT ACA AGC TC-3', reverse, 5'-TGT AGG CAG TAC GGG TCC TC-3' (325 bp); insulin, forward, 5'-GGC TTC TTC TAC ACA CCC A-3', reverse, 5'-CAG TAG TTC TCC AGC TGG TA-3' (182 bp); glucagon, forward, 5'-ATC ATT CCC AGC TTC CCA G-3', reverse, 5'-CGG TTC CTC TTG GTG TTC AT-3' (162 bp); and 18S, forward, 5'-GTA ACC CGT TGA ACC CCA TTC-3', reverse, 5'-CCA TCC AAT CGG TAG TAG CG-3' (151 bp). Real-time RT-PCR analyses were performed with 0.1 µg cDNA using the iQ SYBR Green Supermix kit in Chromo4 Real Time PCR (Bio-Rad Laboratories). Data were normalized to levels of the 18S rRNA subunit. Controls were performed by omitting reverse transcriptase, cDNA, or DNA polymerase (13, 20).

Statistical analysis
Data were expressed as means ± SEM. Statistical significance was determined by unpaired Student’s t test, ANOVA, and Fisher least significant differences post hoc tests using SPSS for Windows (version 11.0; SPSS Inc., Chicago, IL). Differences were considered to be statistically significant when P < 0.05.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
c-KitW-v/+ male mice show high fasting blood glucose levels
The c-Kit mutations are all semidominant with visible differences in fur pigmentation, allowing for precise genotyping of these mice by their distinct coat characteristics. As shown in Fig. 1AGo, no diluted coat color is present in c-Kit+/+ mice; white spotting with slightly diluted pigment is observed in c-KitW-v/+ mice and a complete lack of pigmentation is found in c-KitW-v/W-v mice. The W-v mutation does not affect the levels of c-Kit mRNA and protein in the pancreas (Fig. 1AGo). Although c-KitW-v/W-v mice can survive to maturity, they are infertile and severely anemic. We therefore used c-KitW-v/+ mice for the present study.

To evaluate the effects of the c-KitW-v mutation on glucose metabolism, fasting blood glucose levels were measured in male and female c-Kit+/+ and c-KitW-v/+ mice, at 4, 8, and 16 wk into postnatal life. Among the gender groups, no changes in body weight were noted at 4 wk of age. However, male c-Kit+/+ and c-KitW-v/+ mice showed significant increases in body weight after 8 wk of age when compared with their female counterparts (P < 0.01, Fig. 1BGo). No significant differences in fasting blood glucose levels were noted between the female c-KitW-v/+ and c-Kit+/+ mice at 4, 8, and 16 wk (Fig. 1CGo). Although no difference in fasting blood glucose levels was observed in male c-KitW-v/+ mice at 4 wk of age, these mice exhibited a significant increase in fasting blood glucose levels at 8 and 16 wk when compared with male c-Kit+/+ and female mice groups (P < 0.01, Fig. 1CGo).

c-KitW-v/+ male mice exhibit glucose intolerance and impaired insulin secretion
To further evaluate the effects of the c-KitW-v mutation on glucose metabolism, we performed an IPGTT on these mice. After receiving an ip glucose load, male c-Kit+/+ mice exhibited a blood glucose peak at 15 min after treatment and gradually returned to basal levels by 90 min for mice at 4 wk of age and 120 min for mice at 8–16 wk of age. This pattern of glucose clearance was observed in each of the age groups examined (Fig. 2AGo). However, in 4-wk-old male c-KitW-v/+ mice, an increase in blood glucose levels was noted at 60 (P < 0.01) and 90 (P < 0.05) min after the ip glucose load when compared with 4-wk-old male c-Kit+/+ mice. By 8 wk of age, male c-KitW-v/+ mice were clearly glucose intolerant (Fig. 2AGo). AUCs of the IPGTT graphs were measured to determine corresponding glucose responsiveness (Fig. 2AGo, insets). At 4 wk, male c-KitW-v/+ mice showed no differences in total AUC when compared with male c-Kit+/+ mice (Fig. 2AGo, insets). By 8 and 16 wk, a significant increase in total AUC was noted in male c-KitW-v/+ mice when compared with male c-Kit+/+ mice (P < 0.01, Fig. 2AGo, inset). Interestingly, female c-KitW-v/+ mice showed no differences in the IPGTT at 4 and 8 wk when compared with female c-Kit+/+ mice. A delaying peak, 30 min after receiving the ip glucose load, was observed in female 16-wk-old c-Kit W-v/+ mice (P < 0.05) whereas no differences in total AUC of the IPGTT were noted between female c-KitW-v/+ and c-Kit+/+ mice among all age groups (Fig. 2AGo). Insulin sensitivity determined by IPITT was unaltered in c-KitW-v/+, compared with c-Kit+/+ mice in both male and female groups (Fig. 2BGo).


Figure 2
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  		FIG. 2. A, IPGTT in c-Kit+/+ and c-KitW-v/+ mice at 4, 8, and 16 wk of age. Intolerance to glucose challenge was observed in male c-KitW-v/+ mice at 8 and 16 wk of age. *, P < 0.05; **, P < 0.01 vs. c-Kit+/+ mice (n = 5–19). Glucose responsiveness of the corresponding experimental groups, shown in the small inset, as a measurement of the AUC of the IPGTT graphs with units (millimoles per liter per minute) shown on the y-axis. B, IPITT in c-Kit+/+ and c-KitW-v/+ mice at 8 wk of age showed no changes in insulin sensitivity in all experimental groups (n = 3–4).

 
To assess the effects of the c-KitW-v mutation on insulin secretion in response to administered glucose, glucose-stimulated insulin secretion tests were performed on 8-wk-old male c-KitW-v/+ and c-Kit+/+ mice. Although higher fasting blood glucose levels were observed in male c-KitW-v/+ mice when compared with c-Kit+/+ controls, no significant difference was found in basal plasma insulin levels between the two groups. However, 5 min after glucose stimulation, a 1.2-fold increase in plasma insulin content was noted in both c-KitW-v/+ and c-Kit+/+ mice (Fig. 3AGo). Although there was a further increase in plasma insulin secretion at 35 min after stimulation in c-Kit+/+ mice, a reduction in plasma insulin levels (P < 0.05, Fig. 3AGo) was observed in c-KitW-v/+ mice. After normalization of the glucose-stimulated insulin secretion to fasting blood glucose levels, our results indicated a 19 and 38% increase in insulin secretion at 5 and 35 min, respectively, after glucose stimulation in male c-Kit+/+ mice. However, male c-KitW-v/+ mice showed a 16% increase at 5 min and only a 1% increase after 35 min under the same glucose stimulation conditions. Collectively, these results show that c-KitW-v/+ male mice are impaired in their insulin secretion in response to glucose stimulation.


Figure 3
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  		FIG. 3. Glucose-stimulated insulin secretion tests in 8-wk-old male c-KitW-v/+ and c-Kit+/+ mice and isolated islets. A, Male c-KitW-v/+ mice demonstrate a reduction in plasma insulin release and an increase in plasma glucose 35 min after glucose loading. *, P < 0.05; **, P < 0.01; #, P < 0.05 vs. male c-Kit+/+ mice (n = 5–9). B, Insulin secretion is impaired in isolated islets from male c-KitW-v/+ mice in response to a 22-mM glucose challenge; data are expressed as fold change normalized to basal (2.2 mM glucose) secretion (n = 4). C, Insulin content in isolated islets expressed as nanograms per microgram DNA (n = 6). *, P < 0.04; **, P < 0.01 vs. male c-Kit+/+ mice.

 
We next investigated insulin secretion in isolated islets from 8-wk-old male c-KitW-v/+ and c-Kit+/+ mice. Insulin secretion in response to 22 mM glucose was significantly impaired in the islets of male c-KitW-v/+ mice, showing a 2-fold decrease in insulin secretion after cells were exposed to 22 mM glucose, compared with the male c-Kit+/+ group (P < 0.04, Fig. 3BGo). Total insulin content in isolated islets from 8-wk-old male c-KitW-v/+ mice was significantly lower and demonstrated only 58% of the insulin content measured in the male c-Kit+/+ groups (P < 0.01, Fig. 3CGo).

c-KitW-v/+ male mice display signs of early diabetes with a reduction in β-cell mass
Our results showed that male c-KitW-v/+ mice displayed an early onset of diabetes with noticeable symptoms at 8 wk of age. We therefore performed morphometric analysis to further examine the effect of the c-KitW-v mutation on islet numbers and {alpha}- and β-cell mass in both 4- and 8-wk-old male and female mice. No significant differences in the number of pancreatic islets were noted when experimental groups at 4 (data not shown) and 8 (Fig. 4AGo) wk of age were examined. Interestingly, 4-wk-old male c-KitW-v/+ mice displayed a 50% reduction in β-cell mass when compared with male c-Kit+/+ mice of the same age group (0.24 ± 0.04 vs. 0.48 ± 0.07 mg, P < 0.05) but showed normal fasting blood glucose levels (Fig. 1CGo). A consistent change was observed in 8-wk-old male c-KitW-v/+ mice (P < 0.05, Fig. 4BGo). However, in accordance with our preceding results, c-KitW-v/+ female mice showed no significant reduction in β-cell mass in comparison with c-Kit+/+ female mice at 4 (0.53 ± 0.05 vs. 0.61 ± 0.10 mg) and 8 wk of age (0.69 ± 0.10 mg vs. 0.87 ± 0.17 mg) (Fig. 4BGo). Furthermore, pancreatic insulin content was measured to assess the effects of the c-KitW-v mutation on the ability of the pancreas to produce insulin. Although no significant reduction in insulin product was found in the pancreas of c-KitW-v/+ females at 8 wk age, a significant reduction in insulin content was observed in the c-KitW-v/+ males when compared with the c-Kit+/+ male mice (P < 0.01, Fig. 4DGo). Lastly, we did not detect any changes in {alpha}-cell mass between the genders and among c-Kit+/+ and c-KitW-v/+ mice groups (Fig. 4CGo). These results show a clear reduction in β-cell mass and reduced levels of insulin in male c-KitW-v/+ mice with little change observed in female c-KitW-v/+ mice when compared with their c-Kit+/+ counterparts.


Figure 4
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  		FIG. 4. Morphometric analysis of islet numbers (A), β-cell mass (B), and {alpha}-cell mass (C) in 8-wk-old c-Kit+/+ and c-KitW-v/+ mice (n = 5–6). D, Insulin content in the pancreas of c-Kit+/+ and c-KitW-v/+ mice at 8 wk of age (n = 5–7). *, P < 0.05; **, P < 0.01 vs. male c-Kit+/+ mice; #, P < 0.05; ##, P < 0.01 vs. female c-KitW-v/+ mice.

 
Reduced β-cell proliferation and gene expression in c-KitW-v/+ male mice
The preceding results in this study suggest that the c-KitW-v mutation in male mice is responsible for reduced β-cell mass and insulin production, accounting for the subsequent increase in blood glucose levels. We postulate that c-Kit may be responsible for the proliferation of β-cells within the islet, modulating β-cell mass and therefore ensuring adequate insulin production. To this end, we examined costaining of Ki67 with insulin on pancreatic sections obtained from c-Kit+/+ and c-KitW-v/+ mice at 4 and 8 wk of age. As shown in Fig. 5Go, a significant reduction in Ki67/insulin labeling cells from 4- to 8-wk-old mice in all experimental groups was observed. There is a reduced population of Ki67-positive β-cells in 4-wk-old male c-KitW-v/+ mice, compared with c-Kit+/+ mice, suggesting a decrease in β-cell proliferative capacity in male c-KitW-v/+ mice at this early stage. By 8 wk, this decrease in β-cell proliferation was significant (P < 0.001, Fig. 5BGo). Both female groups showed similar β-cell proliferation rates at 4 and 8 wk of age when compared with male c-Kit+/+ mice (Fig. 5BGo).


Figure 5
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  		FIG. 5. Proliferative capacity of β-cells in c-KitW-v/+ and c-Kit+/+ mice. A, Double-immunofluorescence staining of Ki67 and insulin of 8-wk-old c-KitW-v/+ and c-Kit+/+ mice pancreata; arrows indicate the double-labeling cells. INS, Insulin; DAPI, 4',6'-diamino-2-phenylindole. B, Proliferation rate of β-cells in 4- and 8-wk-old c-KitW-v/+ and c-Kit+/+ mice (n = 5–8). ***, P < 0.001, male c-KitW-v/+ vs. male c-Kit+/+ mice. Scale bar, 25 µm.

 
To evaluate the effects of the c-KitW-v mutation on endocrine cell gene expression, qRT-PCR was performed to measure levels of insulin, glucagon, and Pdx-1 mRNA in 8-wk-old c-Kit+/+ and c-KitW-v/+ male and female mice pancreata and isolated islets. Our results showed no significant differences in pancreatic insulin or glucagon mRNA expression between c-KitW-v/+ and c-Kit+/+ female mice (Fig. 6AGo). In contrast, c-KitW-v/+ males exhibited a significant reduction in pancreatic insulin and Pdx-1 mRNA levels (P < 0.05 and P < 0.04), respectively (Fig. 6AGo). Interestingly, female c-Kit+/+ and c-KitW-v/+ mice expressed higher levels of insulin and glucagon mRNA than male counterparts. In fact, female c-KitW-v/+ mice showed a 7- and 3-fold increase in insulin (P < 0.03) and glucagon mRNA expression, respectively, when compared with male c-KitW-v/+ mice. A 3- and 2-fold increase in insulin and glucagon mRNA expression, respectively, was observed in female c-Kit+/+ mice when compared with male c-Kit+/+ mice (Fig. 6AGo). These differences were also observed in isolated islets (P < 0.01, Fig. 6BGo). Interestingly, Pdx-1 mRNA expression levels did not differ among the c-Kit+/+ gender groups. The decrease in Pdx-1 protein expression in male c-KitW-v/+ mice was further confirmed by immunofluorescence staining and Western blot analysis (P < 0.02, Fig. 7Go).


Figure 6
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  		FIG. 6. Real-time RT-PCR analysis of Pdx-1, insulin, and glucagon mRNA in pancreata (A, n = 5) and isolated islets (B, n = 3–5) from 8-wk-old c-KitW-v/+ and c-Kit+/+ mice. *, P < 0.05, ***, P < 0.001, male c-KitW-v/+ vs. male c-Kit+/+ mice; #, P < 0.05, male c-KitW-v/+ vs. female c-KitWv/+ and c-Kit+/+ mice. &, P < 0.01, male c-KitW-v/+ and c-Kit+/+ mice vs. female c-KitW-v/+ and c-Kit+/+ mice.

 

Figure 7
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  		FIG. 7. Expression of Pdx-1 in 8-wk-old c-KitW-v/+ and c-Kit+/+ mice. A, Double-immunofluorescence staining of Pdx-1 and insulin. INS, Insulin; DAPI, 4',6'-diamino-2-phenylindole. B, Representative blots and quantitative analysis of Pdx-1 expression (n = 4). ***, P < 0.001 vs. c-Kit+/+ male mice. IB, Immunoblot. Scale bar, 25 µm.

 
Delayed onset of abnormal glucose metabolism in c-KitW-v/+ females
To examine whether the impaired glucose metabolism observed in male c-KitW-v/+ mice would occur in females at a later stage, fasting blood glucose levels were measured and glucose tolerance was examined in female c-Kit+/+ and c-KitW-v/+ mice at 40 wk of age. Similar body weights were observed among the c-Kit+/+ and c-KitW-v/+ female mice groups, whereas c-KitW-v/+ female mice displayed a slight increase in fasting blood glucose levels (9.8 ± 3.2 vs. 8.8 ± 2.3 mmol/liter of c-Kit+/+ female mice) (Fig. 8AGo). However, the IPGTT showed that the 40-wk-old female c-KitW-v/+ mice displayed significantly higher blood glucose levels between 15 and 90 min after glucose administration (P < 0.05 or P < 0.01). Lastly, a significantly higher total AUC was detected in female c-KitW-v/+ when compared with female c-Kit+/+ mice at 40 wk of age (P < 0.05, Fig. 8BGo). These results showed that c-KitW-v/+ female mice develop a delayed onset of abnormal glucose metabolism when compared with c-KitW-v/+ male mice.


Figure 8
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  		FIG. 8. Forty-week-old female c-KitW-v/+mice demonstrated glucose intolerance. A, Body weight and fasting blood glucose levels. B, IPGTT studies showed significantly elevated blood glucose levels at all time points after glucose loading in 40-wk-old female c-KitW-v/+ mice, compared with female c-Kit+/+ mice (n = 3–7). *, P < 0.05; **, P < 0.01.

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
Previous studies using the rodent and human fetal pancreas have shown that the c-Kit receptor tyrosine kinase is involved in the proliferation, differentiation and survival of β-cells. However, these investigations did not directly examine the physiological roles of c-Kit in vivo due to a deficient c-Kit receptor. In the present study, we have taken advantage of the mouse mutant, W-v, which expresses a nonfunctional c-Kit receptor and demonstrated that this mutation in c-Kit causes an early onset of diabetes in male mice heterozygous for the mutation. Because c-KitW-v/W-v mice survive for a very short period of time after maturity, they were not included in our present study. However, because the W-v mutation is semidominant, we observed that c-KitW-v/W-v mice display a similar but more severe phenotype than the c-KitW-v/+ mice (data not shown).

Although male c-KitW-v/+ mice showed relatively normal fasting blood glucose levels at 4 wk of age, they displayed a significant increase at 8 and 16 wk. This was further reflected by the fact that high blood glucose levels were observed at all time points post an ip glucose stimulation in 8- and 16-wk-old male c-KitW-v/+ mice. In addition, these mice demonstrated a significant reduction in plasma insulin levels 35 min after glucose stimulation, and their islets showed an impaired insulin secretion response to high glucose. Lastly, by 8 wk, male c-KitW-v/+ mice showed a 50% reduction in pancreatic β-cell mass and insulin content. In support of our observations, Meier et al. (21) showed a 60% β-cell deficiency in minipigs when treated with alloxan, which was associated with high fasting blood glucose levels, glucose intolerance, and low levels of plasma insulin after glucose stimulation. Taken together, these results suggest that the c-KitW-v mutation is responsible for the development of impaired glucose metabolism in male mice between 4 and 8 wk of age. Interestingly, in female mice, intolerance to a high-glucose challenge and changes in pancreatic morphology as well as function occurred much later in life.

Our qRT-PCR and Western blot analyses revealed lower expression levels of Pdx-1 mRNA and protein in male c-KitW-v/+ mice. We previously reported that SCF-stimulated c-Kit receptor activity leads to an increase in Pdx-1 mRNA expression (13). It is well established that Pdx-1 expression is integral to the normal development of the pancreas and maintenance of β-cell function (22, 23, 24, 25). Reduced Pdx-1 mRNA expression in c-KitW-v/+ male mice suggests that not only is the c-Kit receptor required for normal β-cell development and function but that it may be involved in mediating Pdx-1 gene and protein expression.

Dutta et al. (26) reported that Pdx-1 expression is required for β-cell proliferation. In addition, mice containing only one functional allele of Pdx-1 (Pdx-1+/–) show an increase in islet cell apoptosis and decreased β-cell mass (27). Our present findings, that the pancreatic islets of c-KitW-v/+ mice exhibit low levels of Pdx-1, a much lower islet cell proliferation rate, and a decreased β-cell mass, confirm a link between Pdx-1 and islet β-cell development. Moreover, Pdx-1 has been shown to modulate insulin gene expression and to play a role in the regulation of glucose metabolism (28, 29, 30, 31). Thus, the reduced pancreatic insulin expression and glucose intolerance observed in the c-KitW-v/+ mice may also be due to the low levels of Pdx-1 in their pancreatic islets.

It has been shown that phosphorylation of various c-Kit tyrosine residues, due to increased SCF/c-Kit interactions, activates several signaling pathways, including phosphatidylinositol 3-kinase, Janus kinase, MAPK, Src, and phospholipase C-{gamma} in different cell types (32, 33, 34, 35). Rachdi et al. (14) demonstrated that SCF binding to the c-Kit receptor led to the phosphorylation and subsequent activation of ERK1/2 in INS-1 cells. Our recent ex vivo studies using human fetal pancreas showed that exogenous SCF treatment of human fetal islet-epithelial clusters enhanced Pdx-1 expression and cell proliferation and that this process involved increased Akt phosphorylation in a phosphatidylinositol 3-kinase-dependent manner (13). Taken together, these results suggest that c-Kit may be mediating its effects on pancreatic development and function through more than one signaling pathway. However, further investigations are required to elucidate the downstream mechanisms by which c-Kit regulates Pdx-1 expression and pancreatic morphology and function.

Our results indicate that the c-KitW-v mutation does not affect both male and female mice in the same fashion. Although impaired glucose metabolism was observed in 8- and 16-wk-old male c-KitW-v/+ mice, it was not detected in females until 40 wk of age. Morphometric analysis revealed no significant reduction in pancreatic β-cell mass until 8 wk of age, whereas a significant reduction occurred in male c-KitW-v/+ mice by 4 wk. This difference suggests that other mechanisms are capable of compensating for the c-Kit receptor deficiency, thereby protecting female c-KitW-v/+ mice from developing early onset diabetes. The exact mechanism has yet to be characterized, but it is likely that sex hormones play a role. The noted gender differences in glucose metabolism are not unique to our investigation. For instance, male mice with a pttg mutation demonstrated hypoinsulinemia and hyperglycemia (36). Gonadectomy of these mice led to delayed development of diabetes, whereas administration of female sex hormones completely prevented the disease from developing (36). Li et al. (37) also demonstrated that inactivation of the Pdx-1 gene in β-cells resulted in high blood glucose levels and low plasma insulin levels 10 min after glucose stimulation in male mice between 8 and 12 wk of age. However, no changes were observed in female counterparts.

Our mRNA results also reveal gender differences. Pancreatic insulin and glucagon mRNA levels in female c-KitW-v/+ mice are comparable with those in female c-Kit+/+ mice. In addition, there was no significant difference in Pdx-1 mRNA expression between c-KitW-v/+ and c-Kit+/+ females. However, female c-KitW-v/+ and c-Kit+/+ mice demonstrated much higher levels of pancreatic insulin and glucagon mRNA when compared with their male counterparts. Female sex hormones may therefore be involved in stimulating a relative overexpression of these endocrine and β-cell genes. But whether this is responsible for maintaining relatively normal glucose metabolism in c-KitW-v/+ females requires further investigation.

In summary, our study is the first, using the c-KitW-v/+ mice, to demonstrate the physiological and functional effects of the c-KitW-v/+ mutation on the endocrine pancreas in vivo. We show that c-Kit plays an important role in maintaining β-cell mass and glucose metabolism. Although the c-KitW-v mutation is a global one, it is likely that it has direct effects at the level of the developing pancreas because the other affected systems (hematopoiesis, gametogenesis, and melanogenesis) are not closely related with pancreatic endocrine function. Further studies using the c-KitW-v/+ mice should help our understanding of how to restore glucose homeostasis in diabetic patients.


   Acknowledgments
 
The authors are grateful to Dr. C. G. Goodyer (McGill University Health Centre-Montreal Children’s Hospital Research Institute, McGill, Montreal, Quebec, Canada) for her critical comments on the manuscript.


   Footnotes
 
This work was supported by Canadian Institute of Health Research (CIHR) Grant CIHR53227. R.W. is supported by the New Investigator Award from CIHR.

Disclosure Statement: The authors have nothing to disclose.

First Published Online August 2, 2007

1 M.K., F.A., and J.L. contributed equally to this work. Back

Abbreviations: AUC, Area under the curve; IPGTT, ip glucose tolerance test; IPITT, ip insulin tolerance test; Pdx-1, pancreatic duodenal homeobox-1; qRT-PCR, real-time RT-PCR; SCF, stem cell factor.

Received March 23, 2007.

Accepted for publication July 26, 2007.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

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