Induction of human beta-cell proliferation and engraftment using a single G1/S regulatory molecule, cdk6

Abstract

Objective: Most knowledge on human beta-cell cycle control derives from immunoblots of whole human islets, mixtures of beta-cells and non-beta-cells. We explored the presence, subcellular localization, and function of five early G1/S phase molecules-cyclins D1-3 and cdk 4 and 6-in the adult human beta-cell.

Research design and methods: Immunocytochemistry for the five molecules and their relative abilities to drive human beta-cell replication were examined. Human beta-cell replication, cell death, and islet function in vivo were studied in the diabetic NOD-SCID mouse.

Results: Human beta-cells contain easily detectable cdks 4 and 6 and cyclin D3 but variable cyclin D1. Cyclin D2 was only marginally detectable. All five were principally cytoplasmic, not nuclear. Overexpression of the five, alone or in combination, led to variable increases in human beta-cell replication, with the cdk6/cyclin D3 combination being the most robust (15% versus 0.3% in control beta-cells). A single molecule, cdk6, proved to be capable of driving human beta-cell replication in vitro and enhancing human islet engraftment/proliferation in vivo, superior to normal islets and as effectively as the combination of cdk6 plus a D-cyclin.

Conclusions: Human beta-cells contain abundant cdk4, cdk6, and cyclin D3, but variable amounts of cyclin D1. In contrast to rodent beta-cells, they contain little or no detectable cyclin D2. They are primarily cytoplasmic and likely ineffective in basal beta-cell replication. Unexpectedly, cyclin D3 and cdk6 overexpression drives human beta-cell replication most effectively. Most importantly, a single molecule, cdk6, supports robust human beta-cell proliferation and function in vivo.

FIG. 1.

D-cyclins in the human islet and β-cell. A: Western blot for cyclin D₁, D₂, and D₃ in human islets. H1–H3 are three different human islet preparations; H3 was transduced with the adenoviruses overexpressing cyclin D₁ (Ad.D1), cyclin D₂ (Ad.D2), or cyclin D₃ (Ad.D3) to examine cross-reactivity. Each Western blot is representative of three different blots. Islets were extracted between 24 and 48 h of arrival or after infection with adenoviruses. B: Immunoblots for cyclin D₁, D₂, and D₃ in human cell lines and human islets. HK2, SaOS2, and HCK8 are three human cell lines, used for more physiological levels of expression, as compared with the adenoviral overexpression. Each blot is representative of at least three different blots. C: Subcellular localization of cyclin D₁, D₂, and D₃ in human β-cells. Human islets were dispersed as described in Materials and Methods and stained for cyclin D₁, D₂, or D₃ and insulin. Staining with no primary antibody is shown as a negative control. D: Transduced dispersed human islets. Human islets were dispersed and transduced with Ad.D₁, Ad.D₂, or Ad.D₃ and stained for each cyclin D and insulin as described in Materials and Methods. (A high-quality digital representation of this figure is available in the online issue.)

FIG. 2.

Subcellular localization of cdk4 and cdk6 in human β-cells. A: Uninfected dispersed human islets. Human islets were dispersed as described in Materials and Methods and stained for cdk4 or cdk6 and insulin. Staining with primary antibody and blocking peptide is shown as a negative control. B: Dispersed human islet cells transduced with Ad.cdk4 or Ad.cdk6. Human islets were dispersed and transduced with Ad.cdk4 or Ad.cdk6 and stained for cdk4 or cdk6 and insulin as described in Materials and Methods. As can be seen, overexpressed cdk4 and cdk6 are easily detectable, and cdk4 and cdk6 staining is specific. (A high-quality digital representation of this figure is available in the online issue.)

FIG. 3.

Subcellular fractionation of human islets. Human islets were subcellularly fractionated into cytoplasmic fractions (C), as marked with heat shock protein 90 (Hsp90), and nuclear fractions (N) marked with histone 3, and compared to the initial prep of whole (W) human islets. The three components were then immunoblotted for cyclins D1 or D3 (A) or cdk6, cdk4 (B). As can be seen, all four cdk–cyclins are enriched in the cytosolic compartment and not detected in the nuclear compartment. These experiments are representative of a minimum of three human islet preparations.

FIG. 4.

β-cell proliferation in vitro. A: This panel shows examples of isolated whole human islets, embedded in paraffin, sectioned, and stained for insulin (green) and BrdU (red) 72 h after transduction with adenoviruses encoding cdks and D-cyclins. B: Quantification of the BrdU-positive β-cells under each of the conditions. Bars indicate mean ± SEM. n refers to the numbers of human pancreatic islet samples examined. None refers to uninfected islets, LZ refers to Ad.lacZ, D₁ refers to Ad.cyclin D1, D₂ refers to Ad.cyclin D2, D₃ refers to Ad.cyclin D3, C₄ refers to Ad.cdk4, and C₆ refers to cdk6. (A high-quality digital representation of this figure is available in the online issue.)

FIG. 5.

Nuclear translocation of cdk6 in response to adenoviral overexpression of cdk6 alone or in combination with cyclin D1. A: (Upper panel) Human β-cells contain cdk6, but it is largely cytoplasmic. (Middle panel) When cdk6 is overexpressed adenovirally, cdk6 is more abundant and can be observed in the nucleus in some cells. (Lower panel) When cdk6 is adenovirally overexpressed with cyclin D1, cdk6 is more abundant in nuclei. B: Quantification of the percentage of β-cells that contain cdk6 in each of the three conditions shown in A. (A high-quality digital representation of this figure is available in the online issue.)

FIG. 6.

cdk6 alone enhances human islet function in vivo in streptozotocin diabetic NOD-SCID mice. Bars indicate mean ± SEM. A: Mice transplanted with 1,500 IEQ transduced with Ad.lacZ are shown in the black lines, as described in the key within the figure. Mice transplanted with 1,500 IEQ transduced with Ad.cdk6 alone (C₆), with Ad.cyclin D1 alone (D₁), or both Ad.cdk6 and Ad.cyclin D1 (D₁+C₆) are shown in blue, purple, and green, respectively, and compared with 4,000 normal, nontransduced IEQ. The numbers in the key refer to the number of experimental animals in each group. B: Intraperitoneal glucose tolerance testing (IPGTT) in normal (nondiabetic) NOD-SCID mice, diabetic NOD-SCID mice transplanted with 4,000 IEQ human islets, 1,500 human IEQ transduced with Ad.lacZ, or 1,500 IEQ transduced with Ad.cdk6 alone (C₆), with Ad.cyclin D1 alone (D₁) or both Ad.cdk6 and Ad.cyclin D1 (D₁+C₆). Studies were performed 21 days after transplantation. The numbers in parentheses indicate the numbers of animals studied. (A high-quality digital representation of this figure is available in the online issue.)

FIG. 7.

Effects of cdk6, cyclin D1, or cdk6 and cyclin D1 on β-cell proliferation in vivo. A: Proliferation in human β-cells in vivo 28 days after transplantation. Ki-67 is shown in red, and β-cells are shown in green. Human islets grafts were removed at day 28 after transplantation, fixed in 4% paraformaldehyde, embedded in paraffin, and stained for Ki-67. Ki-67 staining in samples of intestine cofixed, coembedded, and cosectioned with the islet grafts were performed as a positive control in the lower right panel. B: Quantification of Ki-67-positive insulin-positive cells as a function of total insulin-positive cells. The numbers shown within the bars indicate the number of Ki-67-positive (red) β-cells and of insulin-positive (green) cells. The numbers below the bars indicate the numbers of animals studied, with two sections per animal. C: cdk6 staining in human β-cells in islet grafts at 28 days posttransplant. Note that cdk6 is still easily visible 28 days posttransplant and that, in many β-cells, it is nuclear as well as cytoplasmic. (A high-quality digital representation of this figure is available in the online issue.)

FIG. 8.

Effects of cdk6, cyclin D1, or cdk6 and cyclin D1 on β-cell death in vivo. A: TUNEL assay was performed on each of the 2–6 fields from 3–4 human islet grafts shown in Fig. 7, 28 days after transplantation. No TUNEL-positive nuclei were observed. TUNEL-positive nuclei are shown in green, and β-cells are shown in red. Examples of TUNEL-positive nuclei are indicated by arrows. TUNEL staining in samples of intestine cofixed, coembedded, and cosectioned with the islet grafts were performed as a positive control in the lower right panel. B: Similar experiments were repeated, but grafts were removed 24 h after transplantation. As can be seen, β-cell apoptosis is similar in the Ad.cdk6-versus Ad.lacZ-transduced islets. (A high-quality digital representation of this figure is available in the online issue.)