Cyclin C stimulates β-cell proliferation in rat and human pancreatic β-cells
Abstract
Activation of pancreatic β-cell proliferation has been proposed as an approach to replace reduced functional β-cell mass in diabetes. Quiescent fibroblasts exit from G0 (quiescence) to G1 through pRb phosphorylation mediated by cyclin C/cdk3 complexes. Overexpression of cyclin D1, D2, D3, or cyclin E induces pancreatic β-cell proliferation. We hypothesized that cyclin C overexpression would induce β-cell proliferation through G0 exit, thus being a potential therapeutic target to recover functional β-cell mass. We used isolated rat and human islets transduced with adenovirus expressing cyclin C. We measured multiple markers of proliferation: [3H]thymidine incorporation, BrdU incorporation and staining, and Ki67 staining. Furthermore, we detected β-cell death by TUNEL, β-cell differentiation by RT-PCR, and β-cell function by glucose-stimulated insulin secretion. Interestingly, we have found that cyclin C increases rat and human β-cell proliferation. This augmented proliferation did not induce β-cell death, dedifferentiation, or dysfunction in rat or human islets. Our results indicate that cyclin C is a potential target for inducing β-cell regeneration.
quiescence is the state of most terminally differentiated cells. This has been confirmed in pancreatic insulin-producing cells, of which nearly all adult β-cells reside in G0 phase of the cell cycle (5, 17). Cyclin C has been proposed as the cell cycle molecule responsible for promoting pRb-dependent G0 exit (20). Ren and Rollins have shown in G0 human fibroblast that cyclin C combined with cdk3 stimulates pRb phosphorylation at S807/811 during the G0–G1 transition, a phosphorylation that is required for cells to exit G0 efficiently (20). It has also been shown that G0–G1 transition in hematopoietic stem cells (HSCs) can be regulated by cyclin C (16). Interestingly, a different role has been shown for cyclin C in postmitotic neurons, where it leads pRb-dependent G0 exit activating the nonhomologous end-joining pathway of DNA repair (NHEJ), pointing that postmitotic cells need to reenter the cell cycle to activate DNA repair (21).
Many cell cycle proteins regulating the G1-S transition have been studied extensively in the pancreatic β-cell, including cdks-4 and -6, d-cyclins, p21, and p27, pRb, p130 (4–8, 11–13, 19, 22). However, the role of cyclin C in the regulation of G0–G1 entrance is unknown in this cell type.
In the search for new therapeutic targets to treat diabetes, one of the proposed approaches is the replacement of lost functional β-cell mass; thus, looking for new molecules that can induce proliferation of β-cells is a key point in diabetes research. On the basis of on previous reports, we hypothesized that cyclin C may be a controllable target for inducing β-cell regeneration. Thus, herein we have investigated the role of cyclin C in β-cell proliferation, death, and function in rat and human islets. Interestingly, we have found that cyclin C overexpression in primary rat and human β-cells induces proliferation without prejudice in β-cell death or function, indicating a potential role for cyclin C in β-cell cycle control and suggesting that it may be a potential target to induce β-cell proliferation.
MATERIALS AND METHODS
Ethical approval.
Experimental procedures were approved by the Animal Care and Use Committee of the University of Cadiz and University of Valladolid in accordance with the Guidelines for Care and Use of Mammals in Research (European Commission Directive 86/609/CEE and Spanish Royal Decree 1201/2005).
INS-1 and rat and human islet cell culture.
The INS-1 832/13 cell line was obtained from Dr. Christopher Newgard of Duke University (14). Cells were grown in RPMI 1640 supplemented with 2 mM l-glutamine 11 mM d-glucose, 10% fetal bovine serum (FBS), 100 U/ml penicillin, 100 μg/ml streptomycin, 10 mM HEPES, 1 mM sodium pyruvate, and 50 μM β-mercaptoethanol. Rat islets were isolated and purified from 2 mo old male Wistar rats as previously reported (7). Human islets were obtained from the Integrated Islet Distribution Program under protocols approved by the University of Michigan. Rat and human islets were grown in RPMI 1640 with 2 mM l-glutamine supplemented with 5.5 mM d-glucose, 10% FBS, 100 U/ml penicillin, and 100 μg/ml streptomycin.
Serum deprivation experiments.
INS-1 cells were serum starved overnight and then exposed to 30 min, 1 h, 2 h, 4 h, and 6 h of medium with serum.
Cytokine experiments.
Rat islets were treated with cytokines for 24 and 48 h. Cytokines were used in the following concentrations: 1,000 U/ml TNFα, 1,000 U/ml IFNγ, and, 50 U/ml IL-1β.
Adenovirus generation and transduction.
The adenoviral vector GFP (which expresses green fluorescent protein under control of the CMV promoter) and the adenoviral vector cyclin C (which expresses human cyclin C protein also under control of the CMV promoter) were produced by the Vector Production Unit in the Center for Animal Biotechnology and Gene Therapy (UPV-CBATEG) at the Universitat Autònoma de Barcelona (Spain). The plasmid containing human cyclin C cDNA was kindly provided by Dr. Barret Rollin's Laboratory, Dana Farber Cancer Institute, Boston, MA.
Rat and human islets were isolated and plated in groups of 400 IEq (islet equivalents). Twenty-four hours later, islets were serum depleted and incubated for 1 h with adenoviral particles at a multiplicity of infection (moi) of 500. Then, medium with adenoviral particles was removed, and transduced islets were incubated in complete medium for 24 h. After this initial incubation, they were incubated in different conditions as detailed in results and the figure legends. For Ki67 experiments in rat islets, groups of 400 IEq were trypsinized for 15 min and then resuspended in 400 μl of medium, and 100 moi of adenovirus was incorporated in a 50-μl drop containing 50,000 cells for 2 h. Afterward, 1 ml was added, and cells were incubated for 48 h.
Western blot.
Transduced islets used for Western blot were incubated for 48 h after transduction. Cells/islets were washed with phosphate-buffered saline (PBS) and lysed in lysis buffer (125 mM Tris, pH 6.8, 2% SDS, 1 mM DTT, and protease/phosphatase inhibitors). The protein lysates were briefly sonicated and centrifuged for 1 min at maximum speed. Proteins were measured by Micro BCA kit (Thermo-Fisher), run on a 12.5% EZ-Run Gel (Fisher Scientific), and then transferred to a PDVF Immobilon-P membrane (Millipore). Blots were incubated with the following antibodies: rabbit anti-cyclin C (Santa Cruz Biotechnology), rabbit anti-actin (Sigma), rabbit anti-Glut2 (Millipore).
β-Cell proliferation: [3H]thymidine incorporation, BrdU incorporation/staining and Ki67 staining.
Twenty-four hours after adenoviral transduction, islets were plated in 24-well plates in 100 IEq groups and cultivated in growth medium without FBS containing [3H]thymidine (1 μCi/well, PerkinElmer) for another 24 h. [3H]thymidine incorporation was corrected for protein levels measured by BCA kit (Thermo-Fisher). Results are expressed as percentage of control.
For BrdU experiments, islets were incubated 24 h in complete medium after transduction and then incubated for other 24 h in serum-free medium containing 10 μM BrdU (Sigma-Aldrich). Afterward, islets were fixed with Bouin's Solution for 1 h and then with formalin until embedded into paraffin blocks. Five- micrometer sections were stained with rat anti-BrdU antibody (Abcam) and with guinea pig anti-insulin antibody (Invitrogen), using anti-guinea pig Alexa fluor 488 and anti-rat Alexa fluor 594 (Invitrogen) as secondary antibodies. Fluorescence images of the sections were acquired using an Olympus BX40 fluorescence axial microscope. The BrdU-positive nuclei of β-cells and the total nuclei of β-cells were counted with the assistance of ImageJ software. At least 500 insulin-positive cells for each preparation were counted.
β-Cell proliferation was also evaluated by the presence of Ki67 in sections of rat/human islets harvested 48 h after transduction, using a specific rabbit monoclonal antibody against Ki67 (Invitrogen) and the same insulin as above. Anti-rabbit Alexa fluor 568 (Invitrogen) was used as secondary antibody. Ki67-positive β-cells and total β-cells were counted with the assistance of the ImageJ software. At least 500 insulin-positive cells were counted per preparation.
Cell death assay.
Islets were incubated for 24 h in complete medium after transduction and then incubated for other 24 h in serum-free medium. Afterward, islets were fixed with Bouin's Solution for 1 h and then with formalin until embedded into paraffin blocks. Five-micrometer sections were double-stained to detect apoptotic β-cells by using the DeadEnd Fluorometric TUNEL (terminal deoxynucleotide transferase-mediated dUTP nick end labeling) histochemistry (System Kit, Promega), following the manufacturer's instructions, and rabbit anti-insulin (Abcam). Anti-rabbit Alexa fluor 568 (Invitrogen) was used as secondary antibody. TUNEL-positive β-cells and total β-cells were counted with the assistance of the ImageJ software. At least 1,000 insulin-positive cells were counted per preparation.
RT-PCR.
Total RNA extraction was performed using the RNeasy Plus Micro kit (Qiagen, Germany) followed by DNAse treatment. cDNA was synthesized using the Transcriptor First Strand cDNA Synthesis kit (Roche, Germany), according to the manufacturer's instructions. Real-time PCR reaction was performed from cDNA using SYBR Premix Ex Taq (Perfect Real Time; Takara Bio, Japan). Analysis of relative gene expression was calculated using the ΔΔCT method, and gene expression levels were normalized to β-actin. Primer sequences are listed in Table 1.
| Gene | Sequence |
|---|---|
| β-Actin-F | 5′-AGC CAT GTA CGT AGC CAT CC-3′ |
| β-Actin-R | 5′-CTC TCA GCT GTG GTG GTG AA-3′ |
| Insulin-F | 5′-CCC AGG CTT TTG TCA AAC AGC A-3′ |
| Insulin-R | 5′-CTC CAG TGC CAA GGT CTG AA-3′ |
| GK-F | 5′-AAG GGA ACA ACA TCG TAG GA-3′ |
| GK-R | 5′-CAT TGG CGG TCT TCA TAG TA-3′ |
| Glut2-F | 5′-TGG GTT CCT TCC AGT TCG-3′ |
| Glut2-R | 5′-AGG CGT CTG GTG TCG TAT G-3′ |
| Kir6.2-F | 5′-GGG CAT TAT CC TGA GGA ATA T-3′ |
| Kir6.2-R | 5′-GAA GGA CAT GGT GAA AAT GAG C-3′ |
| Sur-1-F | 5′-CCA AGG GAA GAT TCA AAT TCA A-3′ |
| Sur-1-R | 5′-GTC CTG TAG GAT GAT GGA CAG G-3′ |
Glucose-stimulated insulin secretion.
Insulin secretion was examined using transduced and control rat/human islets. Islets were plated in cell culture inserts into 24-well plates at a density of 20 IEq/inserted in Hank’s balanced salt solution [HBSS; 114 mM NaCl, 4.7 mM KCl, 1.2 mM KH2PO4, 1.16 mM MgSO4, 20 mM HEPES, 2.5 mM CaCl2, 25.5 mM NaHCO3, and 0.2% bovine serum albumin (essential fatty acid-free), pH 7.2]. Islets were washed three times with 1 ml of HBSS (2.2 mM glucose). Insulin secretion was stimulated using static incubation in 1 ml of HBSS (2.2 mM glucose) for 30 min followed by 30 min in HBSS (22 mM glucose). Secreted insulin from rat islets was measured by ultrasensitive insulin ELISA (Alpco Diagnostics). Secreted insulin from human islets was measured by RIA (Millipore).
Statistics.
Statistical analyses were performed using Student’s t-test when two conditions were compared and using ANOVA when more than two conditions were compared. Data were expressed as means ± SD. P values < 0.05 were considered significant.
RESULTS
Cyclin C expression is associated with β-cell proliferation.
To test whether cyclin C levels were different between β-cells with high proliferation and β-cells in a quiescent state, we measured cyclin C levels by western-blot in islets of 12-wk-old control (db/+) and hyperinsulinemic db/db mice (Fig. 1, A and B) and 4-mo-old Akt mice (AktTg) (Fig. 1, C and D). Young C57BLKS/J db/db mice have shown an almost twofold increase in β-cell proliferation in respect to wild-type β-cells (9, 18). We have found that cyclin C levels were 50% higher in db/db compared with control islet cells (Fig. 1B). On the other hand, AktTg β-cells have shown a twofold increase in proliferation in respect to wild-type β-cells (2). We have found that cyclin C levels were twofold higher in AktTg compared with wild-type islet cells (Fig. 1, C and D).
Fig. 1.Cyclin C expression associates with β-cell proliferation: A: representative Western blot illustrating cyclin C expression in islets of control (db/+) and db/db mice. B: densitometric analysis of western-blots (n = 4) showing the ratio of cyclin C/actin. C: representative western-blot illustrating cyclin C expression in islets of wild-type (WT) and AktTg (Δ4) mice. D: densitometric analysis of western-blots (n = 4) showing the ratio of cyclin C/tubulin. E: representative western-blot illustrating cyclin C expression in INS-1 cells starved of serum and glucose overnight and refed for 30 min to 6 h. F: densitometric analysis of Western blots (n = 4) showing the ratio of cyclin C/actin. G: representative cyclin C Western blot of primary rat islets after cytokine’s mixed treatment (IL-1β, IFNγ and TNFα) for 24 and 48 h, with actin as an internal control. H: densitometric analysis of Western blots (n = 7) showing the ratio of cyclin C/actin at 24 h (gray bar) and 48 h (filled bars) after cytokine treatment. *P < 0.05.
More interestingly, after inducing INS-1 cells to entry in quiescence by overnight deprivation of serum and glucose, cyclin C levels declined. This was reversed after reexposure to FBS and glucose (10% and 11 mM, respectively), with cyclin C being significantly upregulated after 6 h (Fig. 1, E and F).
To test whether cyclin C levels were reduced in conditions of suppression of β-cell proliferation, we tested the antiproliferative effect of proinflammatory cytokines (CK) in cultured β-cells (3). To test whether this effect correlated with a reduction on cyclin C expression, we measured cyclin C levels by Western blot in pancreatic β-cells cultured for 24 and 48 h in the presence or absence of CK (Fig. 1, G and H). We found that cyclin C levels decreased after 24 h, being significantly different at 48 h of CK treatment (Fig. 1H).
Taken together, these experiments suggest an association between cyclin C expression and β-cell proliferation rate, which is upregulated in the setting of higher proliferation and downregulated when proliferation declines.
Cyclin C induces β-cell proliferation.
To test whether increasing cyclin C might enhance β-cell proliferation, we overexpressed cyclin C in rat islets, using an adenoviral vector (Ad.cyclin C) containing human cyclin C cDNA. Ad.cyclin C-transduced rat islets displayed increased cyclin C expression relative to untransduced or Ad.GFP-transduced islets as demonstrated by Western blot using anti-cyclin C antibody, and its expression was dose dependent in respect to the moi (Fig. 2A).
Fig. 2.Cyclin C overexpression induces rat β-cell proliferation. Rat islets were transduced with Ad.cyclin C or Ad.GFP, and proliferation was measured by different techniques. A: representative Western blot showing overexpression of cyclin C in rat islets after 48 h of adenoviral transduction with 100, 200, and 500 moi of adenovirus containing cyclin C or GFP cDNAs. B: cell proliferation was measured by [3H]thymidine incorporation assay (n = 4 in triplicate). C: β-cell proliferation was measured by BrdU incorporation followed by BrdU (red)-insulin (green) staining; scale bar, 50 μm. D: quantification of percentage of BrdU-positive β-cells (n = 5), counting an average of 1,000 β-cells/sample. E: β-cell proliferation was measured by Ki67-insulin staining; scale bar, 50 μm. F: quantification of percentage of Ki67-positive β-cells (n = 4). Representative proliferating β-cells are indicated with white arrows. *P < 0.05.
Overexpression of cyclin D1, D2, D3, or E can induce β-cell proliferation (3, 7, 11). Here, we examined the effect of cyclin C overexpression on rat β-cell proliferation. Cell proliferation induced by cyclin C was assessed by [3H]thymidine incorporation. As shown in Fig. 2B, there was a significant, 1.8-fold increase in proliferation in rat islets transduced with Ad.cyclin C compared with Ad.GFP-transduced islets. To independently confirm this observation, transduced rat islets were cultured with BrdU, fixed, sectioned, and costained with anti-BrdU and anti-insulin antibodies. As shown in Fig. 2, C and D, transduced rat islets revealed a fourfold increase in proliferation as measured by BrdU and insulin costaining. Interestingly, we did not observe increased non-β-cell proliferation in the same conditions (data not shown).
To confirm β-cell proliferation using other cell cycle markers, we studied the expression of Ki67 in dispersed rat islets. This experiment showed that Ki67 was increased 2.5-fold in cyclin C-transduced rat islets compared with Ad.Null, confirming the results observed with BrdU (Fig. 2, E and F). Taken together, these results demonstrate that augmented expression levels of cyclin C increases proliferation in primary cultures of rat islets.
To further investigate the role of cyclin C on human β-cell proliferation, we transduced primary cultures of human islets with Ad.cyclin C and measured [3H]thymidine incorporation. As shown in Fig. 3A, there was augmented expression of cyclin C in human islets transduced with Ad.cyclin C compared with Ad.GFP islets. Ad.cyclin C overexpression induced islet cell proliferation by 120% compared with Ad.GFP islets (Fig. 3B). Interestingly, BrdU data revealed a 1.8-fold increase in β-cell proliferation (Fig. 3, C and D, P = NS), and Ki67 levels showed an ∼140% increase in β-cell proliferation (Fig. 3, E and F, P = NS). Thus, these results demonstrate that cyclin C induces some proliferation in human islets and they partially recapitulate those observed in rat islets.
Fig. 3.Cyclin C overexpression induces human β-cell proliferation. Human islets were transduced with Ad.cyclin C or Ad.GFP, and proliferation was measured by different techniques. A: representative Western blot showing overexpression of cyclin C in human islets after 48 h of adenoviral transduction with 500 moi of adenovirus containing cyclin C or GFP cDNAs. B: β-cell proliferation was measured by [3H]thymidine incorporation assay when transduced with Ad.cyclin C or Ad.GFP (n = 4 in quadruplicate). C and D: β-cell proliferation was measured by BrdU incorporation followed by BrdU-insulin staining when islets were transduced with Ad.cyclin C or Ad.GFP (n = 5); scale bar, 50 μm. E and F: β-cell proliferation was measured by Ki67-insulin staining when transduced with Ad.cyclin C or Ad.GFP (n = 5); scale bar, 25 μm. Representative proliferating β-cells are indicated with white arrows. *P < 0.05.
Human β-cell proliferation experiments have been included in Table 2, containing individual results for each islet donor. We did not observe increased non-β-cell proliferation in human islets (data not shown).
| Donor | Sex | Age, yr | BMI, kg/m2 | Islet Purity, % | SI | Ki67+ β-Cells (%) GFP | Ki67+ β-Cells (%) Cyc C | TUNEL+ β-Cells (%) GFP | TUNEL+ β-Cells (%) Cyc C | [3H]Thymidine Cyc C (fold change) | BrdU+ β-Cells (%) Ad.GFP | BrdU+ β-Cells (%) Ad.Cyc C |
|---|---|---|---|---|---|---|---|---|---|---|---|---|
| Donor 1 | Male | 56 | 30.9 | 95 | 3.78 | 0.25 | 0.39 | 0.24 | 0.25 | 1.17 | 0 | 0.09 |
| Donor 2 | Male | 24 | 29.2 | 97 | 2.1 | 0.26 | 0.35 | 0.18 | 0.43 | 1.07 | 0.11 | 0.09 |
| Donor 3 | Male | 62 | 18.8 | 95 | 7.5 | 0.17 | 0.27 | 0.40 | 0.22 | 0.09 | 0.06 | |
| Donor 4 | Female | 40 | 35.8 | 95 | 7.5 | 0.34 | 0.12 | 0.24 | 0.13 | 1.09 | 0.11 | 0.25 |
| Donor 5 | Male | 54 | 33 | 93 | 7.47 | 0.04 | 0.14 | 0.27 | 0.46 | 1.39 | 0 | 0.04 |
Cyclin C effect on β-cell survival and differentiation.
To test whether cyclin C overexpression would induce deleterious effects into β-cells, we studied β-cell death by TUNEL. Surprisingly, cyclin C decreased β-cell death by 50% compared with control cells in rat islets (Fig. 4, A and B). These data were not recapitulated in human islets, since β-cell death was not affected in human islets transduced with cyclin C compared with control (0.23% ± 0.04 vs. 0.28 ± 0.06). Individual human β-cell death results have been included in Table 1.
Fig. 4.Cyclin C overexpression protects β-cell death in rat islets. Primary rat islets were transduced with 500 moi of Ad.cyclin C or Ad.GFP. A: β-cell death was measured by TUNEL (green)-insulin (red) staining 48 h after transduction; scale bar, 25 μm. B: quantification of percentage of TUNEL-positive β-cells (n = 5), counting an average of 1,000 β-cells/sample. *P < 0.05.
All together, these results indicate that cyclin C expression in rat islets protects β-cell death, and it has no effect on β-cell death in human islets.
Cyclin C partially reverses CK effects on β-cell proliferation and death.
As previously published (3), CK induce a harmful process in the pancreatic β-cell, starting with a decrease in β-cell proliferation followed by induction of β-cell death. Herein, we have recapitulated those experiments showing decreased BrdU incorporation after 24 h of CK treatment (Fig. 5C) that is partially reverted when cyclin C is overexpressed (Fig. 5, B and C, P < 0.05). On the other hand, cyclin C shows protection in basal conditions (Fig. 5D, P < 0.05) and in CK-induced β-cell death after 48 h of CK treatment (Fig. 5, D–F, P = NS). These results nicely correlate with those shown in Fig. 1, E and F, where we have shown decreased cyclin C levels when islets are treated with cyclin C; at the same time, they show a positive effect of cyclin C on β-cell proliferation and death under CK effect.
Fig. 5.Cyclin C partially reverses cytokines effect on β-cell proliferation and death. A–C: effect of cyclin C overexpression on β-cell proliferation of rat islets in basal and cytokine (CK)-induced conditions (n = 4–6). D–F: effect of cyclin C overexpression on β-cell death of rat islets in basal and CK-induced conditions; n = 3–4. *P < 0.05 vs. control without CK; #P < 0.05 vs. control with CK.
Cyclin C does not impair β-cell differentiation or function.
We further investigated other potential adverse effects of higher cyclin C levels on β-cells. To this end, we tested whether cyclin C overexpression led to β-cell dedifferentiation. Thus, we quantified by RT-PCR several β-cell differentiation markers, such as insulin, Glut2, glucokinase, and potassium channel subunits (Sur1, Kir 6.2) in rat islets transduced with Ad.GFP or Ad.cyclin C. Interestingly, β-cell differentiation markers were not affected by cyclin C overexpression (Fig. 6, A–E). We also detected Glut2 expression levels by Western blot (Fig. 6, F and G) and immunostaining (Fig. 6H), detecting no changes in Glut2 expression or localization in islets overexpressing cyclin C compared with controls. To confirm these results, we studied Pdx1/insulin expression by immunostaining. Loss of insulin expression in Pdx1-positive cells would have meant increased dedifferentiation, but in our experiments all insulin-positive cells were Pdx1 positive (Fig. 6I). These results show that cyclin C overexpression induces β-cell proliferation without impairing β-cell differentiation.
Fig. 6.Cyclin C overexpression does not impair pancreatic β-cell differentiation. Primary rat islets were transduced with 500 moi of Ad.cyclin C or Ad.GFP. A–E: real-time PCR analysis using specific primers was performed 48 h after transduction (see materials and methods) for genes involved in β-cell differentiation using total RNA extracted from Ad.cyclin C- and Ad.GFP-transduced primary rat islets (n = 3 in triplicate). Actin was used as a housekeeping gene. F: representative Western blot showing Glut2 expression in islets transduced with Ad.cyclin C (Ad. Cyc) or control (Ad. Null). G: quantification of 4 independent experiments. H: staining of Glut2 (red) and insulin (green) in sections of islets transduced with Ad. Cyc or Ad. Null; scale bar, 25 μm. I: staining of Pdx1 (green) and insulin (red) in sections of islets transduced with Ad. Cyc or Ad. Null; scale bar, 50 μm.
Finally, we studied the effect of cyclin C overexpression on β-cell function. To this end, we performed glucose-stimulated insulin secretion (GSIS) in Ad.cyclin C-transduced islets or controls. As shown in Fig. 7, cyclin C overexpression did not impair insulin secretion after glucose stimulation in rat islets (Fig. 7A) or human islets (Fig. 7B). Therefore, our results on β-cell markers and GSIS support the notion that cyclin C overexpression does not impair β-cell function.
Fig. 7.Cyclin C overexpression does not impair β-cell function. Glucose-stimulated insulin secretion was performed in Ad.cyclin C- or Ad.GFP-transduced rat and human islets. A: groups of 20 rat islet equivalent were incubated for 30 min with 2.2 or 22 mmol/l glucose. Experiments were performed in triplicate (n = 5 in triplicate). B: groups of 20 human islet equivalent were incubated for 30 min with 2.2 or 22 mmol/l glucose. Experiments were performed in quadruplicate (n = 5 in quadruplicate). *P < 0.05.
DISCUSSION
Cyclin C has been proposed as the partner of cdk3 to perform the very first phosphorylation of pRb, required to exit cells from G0 or quiescence (20). Since most pancreatic β-cells are quiescent, it is reasonable to consider whether cyclin C could be a crucial protein for β-cell cycle regulation, i.e., that cyclin C overexpression might lead to β-cell cycle progression, as it has been shown for other cyclins (cyclin D1, D2, D3, or E) (3, 7, 11). This hypothesis was supported by our initial observations showing that cyclin C is elevated in rapidly proliferating db/db and AktTg β-cells and reduced in islet cells showing decreased proliferation in response to CK treatment or INS-1 cells exposed to nutrient deprivation. Using different experimental approaches, we demonstrate for the first time that elevated levels of cyclin C induce proliferation in primary cultures of rat and human pancreatic β-cells. In addition, the current studies show that cyclin C gain of function protects rat β-cell death in basal and CK-induced conditions and that this overexpression has no deleterious effects on β-cell function or differentiation.
It has been shown that cyclin D1, D2, and D3 induce β-cell proliferation through regulation of G1 to S phase (4). Moreover, cyclin E regulates entry in late S phase of the cell cycle in these cells (4). In this way, cyclins are key players in regulating β-cell cycle progression. On the other hand, it has been demonstrated that cyclin C is a critical regulator of the G0/G1 transition in human cells. Thus, Miyata et al. (16) have shown that cyclin C loss of function in human hematopoietic cells resulted in significant increase in quiescent cells without significantly altering the differentiation program, whereas Ren et al. (20) have demonstrated that a gain of function of cyclin C combined with cdk3 is required for human fibroblasts to exit G0 efficiently. Nonetheless, despite our increasing knowledge about the function of cyclin proteins in pancreatic β-cells, it remains an open question whether cyclin C is required for cell cycle progression in quiescent pancreatic β-cells. In this line, it is plausible to propose that exit from G0 in these cells is regulated by cyclin C. Consequently, we have shown that elevated cyclin C levels are associated with increased [3H]thymidine, BrdU incorporation, and Ki67 staining. The current work adds cyclin C to the list of cell cycle modulators capable of driving β-cell proliferation. We have tried to delve deeper into the mechanisms underlying cyclin C-induced proliferation, testing the expression levels of cdk3, cyclin D1, cyclin D2, and pRb phosphorylation and detecting no differences in any of them (data not shown). Further work is warranted to clarify these mechanisms and to determine the extent to which cyclin C drives β-cell proliferation in vivo. This could be tested in animal models with gain or loss of function in β-cells during normal or regenerative conditions.
The changes in β-cell survival obtained by overexpressing cyclin C are interesting and suggest that this cyclin could result in expansion of β-cell mass by inducing proliferation and improving survival. These results are in contrast to other models where high proliferation is associated with increased apoptosis leading to high turnover (1, 10, 13, 15). How cyclin C modulates survival is still unclear, but it is possible that cyclin C could modify responses to oxidative stress (21). We have studied active caspase 3 and p53 expression levels in islets transduced with Ad.cyclin C compared with Ad.GFP, and we have not detected changes (data not shown). In addition, we have observed that survival signals induced by cyclin C are sufficient, at least in part, to inhibit apoptosis induced by CK. Other β-cell stressors such as glucolipotoxicity and endoplasmic reticulum stress will be tested in the near future. Interestingly, this beneficial effect on survival from cyclin C was also accompanied by a lack of adverse effects on insulin secretion. The combination of the changes in survival and the lack of negative effects on insulin secretion make this cyclin C attractive for therapeutic purposes.
In conclusion, we report that cyclin C levels are upregulated in proliferating β-cells and downregulated under conditions of cytokine-mediated stress or nutritional deprivation. Using a gain-of-function approach, we demonstrate that cyclin C leads to β-cell proliferation in rat and human islets.
GRANTS
This work was supported by grants from
DISCLOSURES
No conflicts of interest, financial or otherwise, are declared by the author(s).
AUTHOR CONTRIBUTIONS
Author contributions: M.J.-P., J.F.L.-A., P.V.-P., J.L.M.-A., J.M.-B., B.H.-P., and S.F.-L. performed experiments; M.J.-P., J.F.L.-A., P.V.-P., J.L.M.-A., J.M.-B., B.H.-P., S.F.-L., and I.C.-C. analyzed data; M.J.-P., J.F.L.-A., P.V.-P., G.P., E.B.-M., and I.C.-C. interpreted results of experiments; M.J.-P., J.F.L.-A., P.V.-P., and I.C.-C. prepared figures; M.J.-P., P.V.-P., G.P., E.B.-M., and I.C.-C. drafted manuscript; M.J.-P., J.F.L.-A., J.L.M.-A., J.M.-B., B.H.-P., S.F.-L., G.P., E.B.-M., and I.C.-C. edited and revised manuscript; M.J.-P., J.F.L.-A., P.V.-P., J.L.M.-A., J.M.-B., B.H.-P., S.F.-L., G.P., E.B.-M., and I.C.-C. approved final version of manuscript; G.P. and I.C.-C. conception and design of research.
ACKNOWLEDGMENTS
We thank Dr. Christopher Newgard for sharing the INS-1 832/13 cells and Drs. Shengjun Ren and Barret Rollins for providing the plasmid containing cyclin C cDNA.
REFERENCES
- 1. . Links between apoptosis, proliferation and the cell cycle. Br J Biomed Sci 61: 99–102, 2004.
Crossref | PubMed | ISI | Google Scholar - 2. . Islet β-cell expression of constitutively active Akt1/PKB alpha induces striking hypertrophy, hyperplasia, and hyperinsulinemia. J Clin Invest 108: 1631–1638, 2001.
Crossref | PubMed | ISI | Google Scholar - 3. . Anti-proliferative effect of pro-inflammatory cytokines in cultured β-cells is associated with extracellular signal-regulated kinase 1/2 pathway inhibition: protective role of glucagon-like peptide-1. J Mol Endocrinol 41: 35–44, 2008.
Crossref | PubMed | ISI | Google Scholar - 4. . Molecular control of cell cycle progression in the pancreatic β-cell. Endocr Rev 27: 356–370, 2006.
Crossref | PubMed | ISI | Google Scholar - 5. . Lessons from the first comprehensive molecular characterization of cell cycle control in rodent insulinoma cell lines. Diabetes 57: 3056–3068, 2008.
Crossref | PubMed | ISI | Google Scholar - 6. . The cell cycle inhibitory protein p21cip is not essential for maintaining β-cell cycle arrest or β-cell function in vivo. Diabetes 55: 3271–3278, 2006.
Crossref | PubMed | ISI | Google Scholar - 7. . 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, 2004.
Crossref | PubMed | ISI | Google Scholar - 8. . 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, 2006.
Crossref | PubMed | ISI | Google Scholar - 9. . Characterisation of age-dependent β-cell dynamics in the male db/db mice. PLoS One 8: e82813, 2013.
Crossref | PubMed | ISI | Google Scholar - 10. . Induction of apoptosis in fibroblasts by c-myc protein. Cell 69: 119–128, 1992.
Crossref | PubMed | ISI | Google Scholar - 11. . Induction of human β-cell proliferation and engraftment using a single G1/S regulatory molecule, cdk6. Diabetes 59: 1926–1936, 2010.
Crossref | PubMed | ISI | Google Scholar - 12. . p27 Regulates the transition of β-cells from quiescence to proliferation. Diabetes 55: 2950–2956, 2006.
Crossref | PubMed | ISI | Google Scholar - 13. . The retinoblastoma protein and its homolog p130 regulate the G1/S transition in pancreatic β-cells. Diabetes 58: 1852–1862, 2009.
Crossref | PubMed | ISI | Google Scholar - 14. . Isolation of INS-1-derived cell lines with robust ATP-sensitive K+ channel-dependent and -independent glucose-stimulated insulin secretion. Diabetes 49: 424–430, 2000.
Crossref | PubMed | ISI | Google Scholar - 15. . β-Cell proliferation and apoptosis in the developing normal human pancreas and in hyperinsulinism of infancy. Diabetes 49: 1325–1333, 2000.
Crossref | PubMed | ISI | Google Scholar - 16. . Cyclin C regulates human hematopoietic stem/progenitor cell quiescence. Stem Cells 28: 308–317, 2010.
Crossref | PubMed | ISI | Google Scholar - 17. . Significant human β-cell turnover is limited to the first three decades of life as determined by in vivo thymidine analog incorporation and radiocarbon dating. J Clin Endocrinol Metab 95: E234–239, 2010.
Crossref | PubMed | ISI | Google Scholar - 18. . Reduced proliferation and a high apoptotic frequency of pancreatic β-cells contribute to genetically-determined diabetes susceptibility of db/db BKS mice. Horm Metab Res 43: 306–311, 2011.
Crossref | PubMed | ISI | Google Scholar - 19. . Differential effects of p27 in regulation of β-cell mass during development, neonatal period, and adult life. Diabetes 55: 3520–3528, 2006.
Crossref | PubMed | ISI | Google Scholar - 20. . Cyclin C/cdk3 promotes Rb-dependent G0 exit. Cell 117: 239–251, 2004.
Crossref | PubMed | ISI | Google Scholar - 21. . Cyclin-C-dependent cell-cycle entry is required for activation of non-homologous end joining DNA repair in postmitotic neurons. Cell Death Differ 17: 1189–1198, 2010.
Crossref | PubMed | ISI | Google Scholar - 22. . Essential role of Skp2-mediated p27 degradation in growth and adaptive expansion of pancreatic β-cells. J Clin Invest 117: 2869–2876, 2007.
Crossref | PubMed | ISI | Google Scholar
