To establish which D-cyclins are expressed in the developing pancreas, we carried out RT-PCR on RNA isolated from the developing embryonic pancreas and neonatal pancreas. RT-PCR analysis showed high levels of cyclin D2 expression and lower levels of cyclin D3 in the developing pancreas (Figure A). Immunohistochemistry analysis of D-cyclins in the embryonic pancreas showed that cyclin D2 expression was confined to the nucleus of pancreatic epithelial cells and was absent in differentiated endocrine cells (Figure B). Consistent with the RT-PCR analysis, low levels of cyclin D3 expression was observed in the pancreas during embryonic development (data not shown). Analysis of pancreata from the postnatal period confirmed the complete absence of cyclin D1 expression (Figure C) but showed that the remaining D-cyclins were expressed in a dynamic fashion, often in mutually exclusive cell types. Cyclin D3 expression was restricted to exocrine and ductal cells and was not present in the endocrine cells (Figure D). Cyclin D2, on the other hand, was expressed in the endocrine pancreas as well as the exocrine and ductal tissue (Figure , E and F). Costaining of cyclin D2 with insulin demonstrated that cyclin D2 was expressed in the nuclei of a subset of β cells within islets (Figure , E and F). Cyclin D2 expression was most often seen in larger islets and rarely observed in small clusters of endocrine cells or ductal cells that stain positive for endocrine hormones (Figure F). The proportion of the β cells expressing cyclin D2 increased during the first week after birth and subsequently decreased. As a result, relatively few cyclin D2–positive β cells were visible at the end of the second week (Figure G). Cyclin D2 also decreased in exocrine and ductal tissue, and by postnatal day 14 (P14) these tissues no longer expressed cyclin D2 (Figure H), but continued to express cyclin D3 (data not shown).
Figure 1 Cyclin D2 expression during pancreatic development. We carried out RT-PCR on RNA isolated from embryonic pancreata and stained pancreatic sections from various ages (as indicated) with anti_glucagon (green), anti_insulin (green), and anti_D-cyclin (red; (more ...)
To address whether cyclin D2 was required for endocrine pancreatic development, we analyzed mice in which the cyclin D2 gene had been inactivated by homologous recombination in ES cells. Because cyclin D2 is expressed in pancreatic progenitor cells during embryonic development, we investigated whether cyclin D2 played a role in coordinating the proliferation of progenitor cells with differentiation of endocrine cells. Histological and immunofluorescence analysis revealed no significant differences in islet structure, composition, or endocrine mass in the pancreas from late gestation (embryonic day [E]17.5) cyclin D2–/– embryos compared with WT littermates (data not shown). Thus, cyclin D2 appeared to be dispensable for the formation of the endocrine pancreas during embryonic development.
To determine whether cyclin D2 could play a role in the development of the endocrine pancreas in the postnatal period, we examined the morphology of islets in pancreata from mice 8 weeks after birth. H&E staining revealed a striking reduction in the size of islets in the pancreata from cyclin D2–/– mice as compared with their littermates (Figure , A and B). Immunohistological analysis of insulin and glucagon expression in the pancreatic tissue showed that the smaller islets expressed these hormones (Figure , C and D). No abnormal extraislet staining using a cocktail of islet hormone Ab’s was visible in the pancreata from cyclin D2–/– mice (data not shown). In 6-week-old mice, the mean islet diameter of WT mice was 202 ± 13 μm versus 76.5 ± 4 μm for the cyclin D2–/– mice (P < 0.0001). The islets in cyclin D2–/– mice retained the characteristic distribution of endocrine cells with β cells forming the core and α cells at the periphery forming the mantle. No necrosis or fibrosis was apparent in the cyclin D2–/– pancreata and the size and weight of these pancreata were similar to the WT littermates. The size of individual β cells did not appear to differ in the cyclin D2–/– and WT littermates, indicating that the reduced islet size was likely due to a decrease in the number of β cells. Because the number of endocrine cells depends on the balance of neogenesis, replication, and apoptosis of endocrine cells, we considered whether any of these mechanisms were affected by the absence of cyclin D2.
Figure 2 Islet morphology and neogenesis of endocrine cells in WT and cyclin D2–/– mice. We stained pancreatic sections from 6-week-old WT and cyclin D2–/– (_/_) littermates with anti_glucagon and anti_insulin Ab’s. We estimated (more ...)
A number of observations, including prevalence of hormone-positive ductal cells as well as the close proximity of islets to ducts, have led to the idea that endocrine cell differentiation occurs in the ducts (12
). In WT neonates, hormone-positive staining of ductal cells was rarely observed right after birth, but was more frequently observed by the end of the first week of postnatal development (Figure E). In cyclin D2–/–
P7 mice, hormone-positive ductal cells were clearly visible at roughly similar frequencies as in WT littermates (Figure F). In addition, the distribution of extraislet cells with immunoreactive insulin was similar in WT and cyclin D2–/–
mice. These results indicate that the frequency of hormone-positive ductal cells was unaffected by the absence of cyclin D2
. We also did not observe any difference in the levels of apoptosis in the endocrine pancreas in WT and cyclin D2–/–
mice (data not shown).
We examined the replicative state of β cells during the postnatal period by assessing their ability to incorporate BrdU. BrdU, a thymidine analogue, is incorporated in the nucleus of cycling cells during the S-phase of the cycle. Throughout the first week after birth, pancreata from WT neonates showed a large number of cells within islets stained for BrdU. Costaining with insulin confirmed that a large proportion of β cells underwent replication in the early postnatal period (Figure A). Strikingly, no incorporation of BrdU was observed in β cells in the pancreata from cyclin D2–/– littermates (Figure B). Comparison of the replicative index in WT versus cyclin D2–/– P4 neonates revealed a marked defect in β cell replication in cyclin D2–/– mice (9.2% versus 0%, P < 0.001). To test whether cyclin D2 plays a role in the replication of other endocrine cells, we examined in these pancreata whether glucagon-stained cells also incorporated BrdU. In pancreata from WT neonates, a few glucagon-positive cells showed incorporation of BrdU, indicating that α cells, in addition to β cells, replicate and expand during early postnatal development (Figure C). Again, no incorporation of BrdU was observed in α cells in the pancreata from cyclin D2–/– littermates (Figure D). The replication defect in cyclin D2–/– mice was restricted to the endocrine pancreas as exocrine and ductal cells that incorporated BrdU were readily visible. To further characterize replication in the pancreata of cyclin D2–/– mice, we examined the incorporation of BrdU in exocrine tissue identified by amylase staining. No difference in the BrdU incorporation in exocrine tissue was observed in the cyclin D2–/– pancreata compared with those from WT littermates (Figure , E and F).
Figure 3 Incorporation of BrdU during postnatal pancreatic development of WT and cyclin D2–/– mice. BrdU was injected in 4- and 7-day-old mice 2 hours before being sacrificed. (A and B) Sections from pancreata costained with anti_insulin and anti_BrdU (more ...)
We next examined whether the highly selective defect in endocrine cell replication in the absence of cyclin D2 was due to the inability of endocrine cells to upregulate the remaining D-cyclins. We compared the expression of cyclin D1 and D3 in the pancreata of neonatal WT and cyclin D2–/– littermates. As shown earlier, in WT P4 mice, cyclin D3 was expressed in the exocrine and ductal tissue, but absent from endocrine tissue (Figure A). In cyclin D2–/– littermates, the expression of cyclin D3 in the exocrine and ductal tissue was similar to the WT mice. Significantly, costaining with insulin demonstrated that cyclin D3 expression is not detectable in β cells from cyclin D2–/– mice (Figure B). We also failed to observe any cyclin D1 expression in β cells of the pancreas from P4 cyclin D2–/– mice (Figure C). We next examined the expression of cyclin D1 and D3 in cyclin D2–/– mice at the end of the postnatal remodeling phase of the endocrine pancreas. Pancreatic sections from P14 WT mice did not stain for cyclin D1 (Figure D). In the cyclin D2–/– littermates, however, cyclin D1 expression was clearly detectable in the endocrine pancreas. Costaining with glucagon revealed the cyclin D1–expressing cells in the pancreata from cyclin D2–/– mice were restricted to the core of the islets (Figure E), and insulin staining demonstrated that cyclin D1 expression was expressed in the nuclei of β cells (Figure F). Cyclin D3 expression continued to be detected in the exocrine and ductal tissue in P14 WT and cyclin D2–/– mice, but not in endocrine cells (data not shown).
Figure 4 Expression of cyclin D1 and D3 in WT and cyclin D2–/– mice during postnatal development. (A and B) Sections from pancreata of 4-day-old WT and cyclin D2–/– mice were stained with anti_cyclin D3 and anti_insulin Ab’s. (more ...)
Because β cell replication is an important parameter that regulates β cell mass, we quantified the total β cell mass during the 2-week period of postnatal development. At P4, the β cell mass in the pancreas of cyclin D2–/– mice was similar to its WT littermates (WT, 0.39 ± 0.02 mg, versus cyclin D2–/–, 0.36 ± 0.01 mg). The β cell mass subsequently increased rapidly during postnatal development, and a 4-fold increase in β cell mass was apparent in P14 WT mice (Figure A). By contrast, no significant increase in the β cell mass was apparent in the cyclin D2–/– littermates. Consequently, by P14, the total β cell mass of cyclin D2–/– mice is about 30% of that of their WT littermates (cyclin D2–/–, 0.5 ± 0.05 mg, versus WT, 1.5 ± 0.16 mg; P < 0.05). This lack of increase in mass was specific to the endocrine pancreas, because the total weight of an individual pancreas from cyclin D2–/– mice did not differ significantly from their WT littermates throughout postnatal development. This is consistent with the observation that exocrine tissue, which makes up 98% of the pancreas, was unaffected in the absence of cyclin D2. Moreover, the cyclin D2–/– mice did not differ from their WT littermates in body weight or size (Figure B). Thus, unlike WT mice, the increase in body weight of cyclin D2–/– mice was not matched by the increment in β cell mass (Figure C).
Figure 5 β cell mass in WT and cyclin D2–/– mice during postnatal development. (A) The β cell mass per pancreas was estimated as the product of the relative cross-sectional area of β cells (determined by quantification of (more ...)
To test whether the decrease in the relative β cell mass of cyclin D2–/– mice resulted in altered serum insulin levels and abnormal glucose homeostasis, we measured serum insulin levels and performed glucose tolerance tests (GTTs) in WT and cyclin D2–/– mice. The serum insulin levels after overnight fasting were similar in WT and cyclin D2–/– mice. After glucose challenge, however, serum insulin levels doubled in WT mice but only increased slightly in cyclin D2–/– mice (Figure A). The cyclin D2–/– mice had fasting blood glucose levels that were consistently elevated when compared with the WT mice. The cyclin D2–/– mice also showed a decreased ability to clear glucose from the blood following intraperitoneal glucose injection. WT mice peaked at levels between 140 and 160 mg/dl 15 minutes after injection and reached baseline by the end of the 90-minute testing period (Figure B). In contrast, cyclin D2–/– mice had elevated blood glucose levels (300–350 mg/dl) within 15 minutes of injections. Moreover, the blood glucose levels failed to return to baseline levels during the testing period and remained in the range of 150–250 mg/dl 120 minutes after the injection (data not shown).
Figure 6 Cyclin D2 mutant mice have decreased plasma insulin levels and impaired glucose tolerance. (A) Plasma insulin levels in WT and cyclin D2–/– mice following overnight fasting and 30 minutes after glucose challenge (2 g/kg body weight). Results (more ...)