Critical role of c-Kit in beta cell function: increased insulin secretion and protection against diabetes in a mouse model
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The receptor tyrosine kinase, c-Kit, and its ligand, stem cell factor, control a variety of cellular processes, including pancreatic beta cell survival and differentiation as revealed in c-Kit Wv mice, which have a point mutation in the c-Kit allele leading to loss of kinase activity and develop diabetes. The present study further investigated the intrinsic role of c-Kit in beta cells, especially the underlying mechanisms that influence beta cell function.
We generated a novel transgenic mouse model with c-KIT overexpression specifically in beta cells (c-KitβTg) to further examine the physiological and functional roles of c-Kit in beta cells. Isolated islets from these mice were used to investigate the underlying molecular pathway of c-Kit in beta cells. We also characterised the ability of c-Kit to protect animals from high-fat-diet-induced diabetes, as well as to rescue c-Kit Wv mice from early onset of diabetes.
c-KitβTg mice exhibited improved beta cell function, with significantly improved insulin secretion, and increased beta cell mass and proliferation in response to high-fat-diet-induced diabetes. c-KitβTg islets exhibited upregulation of: (1) insulin receptor and IRSs; (2) Akt and glycogen synthase kinase 3β phosphorylation; and (3) transcription factors important for islet function. c-KIT overexpression in beta cells also rescued diabetes observed in c-Kit Wv mice.
These findings demonstrate that c-Kit plays a direct protective role in beta cells, by regulating glucose metabolism and beta cell function. c-Kit may therefore represent a novel target for treating diabetes.
KeywordsBeta cell function Beta cell-specific c-KIT transgenic mice c-Kit Wv mutation High-fat-diet-induced diabetes Insulin secretion
- c-KitβTg mice
Transgenic mouse model with c-KIT overexpression specifically in beta cells
Enhanced green fluorescent protein
Glucose-stimulated insulin secretion
Glycogen synthase kinase 3β
Intraperitoneal glucose tolerance test
Intraperitoneal insulin tolerance test
v-Maf musculoaponeurotic fibrosarcoma oncogene family, protein A (avian)
Pancreatic and duodenal homeobox 1
Rat insulin promoter
Stem cell factor
Therapeutic strategies aimed at repopulating insulin-producing cells show great potential for restoring glycaemia in diabetes. Extensive studies have focused on ways to facilitate the differentiation of progenitor cells into beta cells [1, 2, 3], and to maintain their viability and function [4, 5]. It has been shown that the haematopoietic stem cell marker c-Kit is important in the development and function of islets of Langerhans, especially in support of beta cell proliferation, maturation and survival [6, 7, 8, 9, 10, 11, 12]. Upon binding to the stem cell factor (SCF), c-Kit undergoes dimerisation and autophosphorylation, followed by the recruitment of downstream signalling molecules to induce subsequent cell proliferation, differentiation, survival and migration [13, 14].
c-Kit is found in fetal and adult rodent pancreatic islets [6, 7, 8, 10]. We have demonstrated that human and rat fetal pancreatic ductal epithelial cells producing c-Kit display high proliferation and level of SCF [11, 12, 15]. After pancreatic duct ligation in the rat, c-Kit is activated in ductal cells during islet cell neogenesis, along with an increase of pancreatic and duodenal homeobox 1 (PDX1) levels . Increased c-Kit and PDX1 abundance is also observed in islets from pancreases of streptozotocin-induced diabetic rats, suggesting that c-Kit is involved in beta cell regeneration .
Manipulation of rat islets in cell culture has further revealed that c-Kit-enriched cells can give rise to new beta cells that secrete insulin in a glucose-responsive fashion . Fetal rat islets treated with SCF had a significant increase in insulin levels and DNA content . Furthermore, INS-1 cells responded to SCF with increased cell proliferation . Downregulation of c-KIT (also known as KIT) expression in human islet–epithelial clusters using small interfering RNA leads to significantly reduced mRNA and protein levels of PDX1 and insulin in conjunction with decreased cell proliferation, as well as increased cell death . These studies reveal a remarkable correlation between the functions of c-Kit and enhanced beta cell development and function.
Homozygous c-Kit-null (c-Kit W/W ) mutant mice, display relatively normal islet morphology, but die shortly after birth and are not available for further functional studies . We have previously characterised c-Kit Wv mice, which have a point mutation in the c-Kit allele, disrupting receptor function. These mice exhibit a loss of beta cell mass and proliferation, resulting in early onset of diabetes . In the present study, we describe a novel transgenic mouse model with c-KIT overexpression specifically in beta cells (c-KitβTg), which we used to investigate the underlying mechanism of c-Kit activity in beta cells, and to further delineate the physiological and functional role of c-Kit in normal, high-fat diet (HFD)-induced diabetic and c-Kit Wv mice.
Generation and maintenance of c-KitβTg mice
Human c-KIT cDNA (2.9 kb pairs) followed by the IRES2-enhanced green fluorescence protein (eGFP) (Clontech, Palo Alto, CA, USA) sequence was inserted into the pKS/rat insulin promoter (RIP) plasmid  to generate the transgene. Transgenic mice (c-KitβTg) were generated using C57BL6/J embryos and identified by PCR  using the primers globin 5 and c-Kit, and further confirmed by a second set of primers, eGFP3 and globin 3 (Electronic supplementary material [ESM] Table 1). Five RIP–c-KIT transgenic founders were obtained and bred with C57BL/6J mice (Jackson Laboratory, Bar Harbor, ME, USA) to establish independent mouse lines. Initial characterisation revealed that the mice displayed similar, if not identical, patterns of transgene expression and phenotype that eliminate any positional effect due to the location of transgene integration. We therefore used offspring from two independent transgenic lines for subsequent detailed analyses. All mice had free access to standard diet. The protocol used was approved by the University of Western Ontario Animal User Subcommittee in accordance with the guidelines of the Canadian Council of Animal Care.
Body weight, food intake and in vivo metabolic studies
Body weight, blood glucose levels, intraperitoneal glucose tolerance tests (IPGTT) and intraperitoneal insulin tolerance tests (IPITT) were performed using c-KitβTg mice and their wild-type littermates from 4 to 40 weeks of age as described previously [20, 23]. Food intake was monitored at 6 weeks of age for a 2 week period. Blood glucose levels were examined under non-fasting, and after 4 h and overnight fasting (16 h) with free access to water. For the IPGTT and IPITT, an intraperitoneal injection of glucose (d-(+)-glucose; dextrose; Sigma, St Louis, MO, USA) at a dosage of 2 mg/g body weight or of human insulin (Humalin; Eli Lilly, Toronto, ON, Canada) at 1 U/kg body weight was administered and blood glucose levels were examined. The AUC was used to quantify responsiveness [20, 23].
Generation of HFD-induced diabetes and c-KitβTg:Wv mouse models
The HFD study was initiated at 6 weeks of age, with c-KitβTg and wild-type male mice receiving HFD chow (D12492; Research Diets, New Brunswick, NJ, USA) for 4 weeks, followed by in vivo metabolic studies [24, 25]. Breeding c-KitβTg with c-Kit Wv mice yielded four different mouse genotypes: (1) wild-type; (2) c-KitβTg; (3) c-Kit Wv and (4) c-KitβTg:Wv. Presence of the c-Kit Wv allele was identified by the animal’s characteristic fur pigmentation ; the c-KitβTg allele was identified by PCR. In vivo metabolic studies were performed on male mice groups at 8 weeks of age.
INS-1 cell culture
INS-1 (832/13) cells were cultured in RPMI 1640 containing 10% FBS vol./vol., as described previously . For the exogenous SCF study, INS-1 cells were cultured in RPMI 1640 plus 1% (wt/vol.) BSA, and treated for 24 h with SCF (50 ng/ml; ID Laboratory, London, ON, Canada), SCF plus wortmannin (100 nmol/l; Sigma) or non-SCF (control) . Cells were collected for immunofluorescence staining and protein extraction. Cell proliferation was examined using the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay and Ki67 staining . Three different cell passages were used for each set of experiments, representing n = 3.
In vivo and ex vivo glucose-stimulated insulin secretion assay and insulin ELISA
For the in vivo glucose-stimulated insulin secretion (GSIS) assay, blood samples were collected following 4 h of fasting (0 min), and at 5 and 35 min after glucose loading [20, 23]. For the ex vivo GSIS assay, freshly isolated islets from mice were hand-picked and incubated for 1 h with RPMI 1640 plus 0.5% (wt/vol.) BSA containing 2.2 or 22 mmol/l glucose . Insulin secretion was measured using an ultrasensitive (mouse) insulin ELISA (Alpco, Salem, NH, USA). A static GSIS index was calculated [20, 26]. The insulin content of isolated islets was measured and expressed as nanograms per microgram DNA.
Immunofluorescence and morphometric analyses
Pancreases were fixed in 4% (wt/vol.) paraformaldehyde, and sections prepared from the entire length of the pancreas and stained with primary antibodies of dilutions as listed (ESM Table 2). Quantitative evaluations of alpha cell and beta cell mass were performed using Openlab image software (Improvision, Lexington, MA, USA) [20, 23]. Beta cell proliferation and levels of different transcription factors were determined by double immunofluorescence staining and quantification from at least 12 random islets per pancreatic section [20, 23].
RNA extraction and real-time RT-PCR
RNA was extracted from isolated islets of c-KitβTg and wild-type mice with or without HFD using a kit (miRNeasy; Qiagen, Germantown, MD, USA) . Sequences of PCR primers are listed in ESM Table 3 . Real-time PCR analyses were performed using a kit (iQ SYBR Green Supermix; Bio-Rad Laboratories, Mississauga, ON, Canada). Relative levels of gene expression were calculated and normalised to the internal gene, 18S rRNA, with at least four repeats per age per experimental group [20, 23].
Protein extraction and Western blot analysis
Islet proteins from c-KitβTg and wild-type pancreases were extracted in NP-40 lysis buffer [20, 26]. An equal amount of protein from each group was fractionated by 10% (wt/vol.) SDS-PAGE, transferred on to a nitrocellulose membrane (Bio-Rad) and incubated with primary antibodies as listed (ESM Table 2). Proteins were detected using western blot detection reagents (ECL-Plus; Perkin Elmer, Wellesley, MA, USA) and imaged using Versadoc Imaging System (Bio-Rad). Bands were densitometrically quantified by Image lab software (Bio-Rad).
Data are expressed as means ± SEM. Statistical significance was determined by unpaired Student’s t test or ANOVA followed by Fisher’s least significant differences post-hoc test. Differences were considered to be statistically significant at p < 0.05.
Generation of c-KitβTg mouse model
Improved glucose tolerance and insulin secretion in c-KitβTg mice
Increased islet transcription factors, beta cell mass and proliferation in c-KitβTg mice
Increased abundance of insulin receptor, phosphorylated IRS1/2 and their downstream signalling molecules in the islets of c-KitβTg mice
c-KitβTg mice tolerated HFD-induced diabetes
c-Kit Wv mice with specific overexpression of c-Kit in beta cells displayed normal glucose metabolism
Here, we demonstrated that c-KIT overexpression in beta cells confers improved glucose metabolism by enhancing insulin secretion, and increasing beta cell mass and proliferation, probably through activation of the PI3K–Akt signalling pathway. When c-KitβTg mice were subjected to a HFD, they displayed resistance to HFD-induced glucose intolerance and preserved beta cell function relative to wild-type littermates. Moreover, c-KitβTg mice were protected against early onset of diabetes. These results clearly indicate that c-Kit is intrinsic to beta cell function and proliferation. This effect is mediated by the regulation of key beta cell transcription factors (e.g. PDX1 and v-maf musculoaponeurotic fibrosarcoma oncogene family, protein A (avian) [MAFA]) and possibly through interaction with the insulin receptor to activate downstream PI3K–Akt signalling.
Compared with wild-type mice, c-KitβTg mice had significantly lower overnight fasting blood glucose levels, improved glucose tolerance and enhanced GSIS. The improvement in glucose metabolism of c-KitβTg mice was associated with an increase in beta cell mass and proliferation, as well as in Glut2 and Glp1r expression. These results corroborate our previous finding of glucose intolerance in c-Kit Wv mice . We monitored the c-KitβTg mice up to 40 weeks of age and did not detect any abnormal cell growth or islet tumours, indicating that beta cell-specific c-KIT overexpression does not lead to malignancy. We detected a significant increase in Scf mRNA, but only a modest increase in the corresponding protein. This is likely to be due to our western blotting technique, which could only detect membrane-associated, not soluble, SCF. Nevertheless, the concentration of SCF may be in excess and is sufficient to interact with the increased number of c-Kit receptors (∼25%) to transduce markedly enhanced intracellular signals in c-KitβTg mice. Our data indicate that c-Kit overabundance in beta cells enhances glucose tolerance and beta cell function in males, while the effect is less significant in female mice. These sex-related differences in glucose metabolism are consistent with our previous results using c-Kit Wv mice and conditional β1 integrin knockout mice [20, 23], and have also been described in other mouse models [28, 29]. We observed that isolated islets from female c-KitβTg mice had significantly enhanced insulin secretion in response to a high glucose challenge compared with wild-type females, indicating that the sex-related differences may involve other pathways, such as the contribution of oestrogen to glucose homeostasis in female rodents.
Significantly increased Pdx1 mRNA and protein abundance was observed in c-KitβTg mice. We previously reported that SCF-stimulated c-Kit receptor activity leads to increased PDX1 mRNA expression in human fetal islet–epithelial clusters , while c-Kit Wv mice showed a significant reduction in Pdx1 expression in islets . It is well documented that PDX1 is integral to normal pancreas development and beta cell function . In addition to beta cell proliferation , Pdx1 expression is also required for modulation of insulin gene expression and glucose metabolism . Our results showed that c-KitβTg islets exhibited high PDX1 levels and had increased beta cell proliferation and mass, confirming a correlation between PDX1 and islet beta cell replication. Indeed, the enhanced islet insulin secretion in response to a high glucose challenge and the improved glucose tolerance observed in c-KitβTg mice may also be due to increased islet PDX1 abundance. Interestingly, significant upregulation of Mafa mRNA was observed in both sexes of c-KitβTg mice. MAFA binds to the C1 element of the insulin gene to modulate insulin gene transcription and enhance beta cell maturation . Mafa-null mice had a defect only in adult islet architecture and beta cell activity, while MAFA overproduction enhanced beta cell insulin biosynthesis and secretion through upregulation of important beta cell genes, including Pdx1, Neurod1, Nkx6-1 and Glp1r . Moreover, overexpression of MAFA in neonatal rat beta cells led to enhanced glucose-responsive insulin secretion and beta cell maturation . Thus, improved glucose metabolism and insulin secretion in c-KitβTg mice may be due to increased c-Kit receptor stimulation of islet Pdx1 and Mafa expression.
The molecular mechanisms associated with the phenotypic changes observed in c-KitβTg mice include significant upregulation of phospho-Akt and phospho-GSK3β in beta cells. Our previous in vitro study on human fetal islet–epithelial clusters demonstrated that increased c-Kit × SCF interactions resulted in upregulation of PI3K–Akt, but not of mitogen-activated protein kinase (MAPK)–extracellular signal-regulated kinase (ERK) pathway signalling . In the current study, we also observed significant increases in insulin receptor and phospho-IRS1/2 levels, in addition to the increase in Akt and GSK3β phosphorylation together with upregulation of cyclin D1 in islets isolated from c-KitβTg mice. These results suggest the following possible mechanisms, whereby c-Kit might stimulate beta cell function and proliferation: (1) via direct activation of the Akt–GSK3β–cyclin D1 pathways [15, 36]; (2) via direct or indirect interaction between c-Kit and the insulin receptor and IRSs to establish cross-talk; and/or (3) by positive feedback of the insulin receptor via PI3K–Akt signalling in association with PDX1- and Mafa-induced insulin secretion. Islets of beta cell-specific Gsk3β ablated mice had increased beta cell mass with lower fasting blood glucose, along with improved glucose tolerance and GSIS . Furthermore, beta cell-specific Gsk3β knockout mice had increased islet IRS-1 and -2 levels with significantly improved beta cell function , which is in agreement with our observations. It has been reported that insulin secreted by pancreatic beta cells positively regulates its own biosynthesis by enhancing insulin gene transcription in an autocrine manner via the beta cell insulin receptor and downstream signalling pathways [38, 39]. Specific knockout of Insr in pancreatic beta cells results in defective insulin secretion, similar to that observed in type 2 diabetes . Taken together, these results suggest that Akt–GSK3β–cyclin D1 signalling downstream of c-Kit is essential for beta cell function.
HFD treatment is detrimental to beta cell function and insulin sensitivity in mice [24, 41], and leads to impaired glucose tolerance due to insulin resistance and insufficient beta cell insulin secretion [24, 41, 42, 43]. While c-KitβTg and wild-type mice maintained on a HFD showed similar food intake and weight gain, significantly less fat pad formation was observed in the former after 4 weeks on the HFD. Importantly, c-KitβTg HFD mice exhibited significantly improved glucose tolerance and GSIS, supporting the notion that c-Kit has a direct effect on beta cell function. Islets of c-KitβTg HFD mice also showed significantly increased levels of insulin receptor and insulin signals, suggesting a possible secondary mechanism, whereby c-Kit stimulates beta cell function by cross-talking to the insulin receptor via PI3K–Akt signalling. Therefore, overabundance of c-Kit in beta cells plays a primary role by increasing beta cell mass and proliferation, as well as a secondary role by increasing insulin secretion via upregulation of the insulin receptor through the PI3K–Akt signalling pathway, which enables c-KitβTg HFD mice to tolerate HFD-induced diabetes.
Finally, we bred c-KitβTg with c-Kit Wv mice to determine whether c-KIT overexpression could prevent beta cell dysfunction in c-Kit Wv mice. Our results showed that c-KitβTg:Wv mice displayed normal fasting glycaemia and glucose tolerance, as well as enhanced glucose-induced insulin secretion. This marked improvement in glucose metabolism in c-KitβTg:Wv mice provides direct evidence of a primary effect of the c-Kit receptor on beta cell function.
In summary, we showed that c-KIT overexpression in beta cells led to improved beta cell proliferation and function, and protected mice from HFD-induced diabetes. Furthermore, beta cell-specific overexpression of c-KIT was able to prevent beta cell defects in c-Kit Wv mice. This study provides direct evidence to support the notion that c-Kit plays a primary physiological role in beta cells, and thus may help efforts to develop gene and cell therapeutic schemes for patients with diabetes.
This work was supported by grants from the Canadian Institutes of Health Research (CIHR, grant number MOP 89800).
Duality of interest
The authors declare that there is no duality of interest associated with this manuscript.
ZCF and JL contributed to the acquisition of data, data analysis and interpretation, manuscript drafting and final approval of the manuscript. BAT contributed to the data analysis and interpretation, manuscript drafting and final approval of the manuscript. MR contributed to analysis and interpretation of data, revising the article, and final approval of the manuscript. SPY contributed to the design of the transgenic model, analysis and interpretation of data, the provision of study materials, and the critical revision and final approval of the manuscript. RW contributed to the conception and design of the study, the collection and assembly of data, data analysis and interpretation, manuscript writing and final approval of the manuscript.
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