Proliferation is the major component for maintenance of ␤-cell mass in adult animals. Activation of phosphoinositide 3-kinase/Akt-kinase pathway is a critical regulator of ␤-cell mass. Pancreatic ␤-cell overexpression of constitutively active Akt in mice (caAkt Tg ) resulted in marked expansion of ␤-cell mass by increase in ␤-cell proliferation and size. The current studies provide new insights into the molecular mechanisms involved in ␤-cell proliferation by Akt. Proliferation of ␤-cells in caAkt Tg was associated with increased cyclin D1, cyclin D2, and p21 levels and cyclindependent kinase-4 (cdk4) activity. To determine the role of cdk4 in ␤-cell proliferation induced by Akt, we generated caAkt Tg mice that were homozygous, heterozygous, or nullizygous for cdk4. The results of these studies showed that deletion of one cdk4 allele significantly reduced ␤-cell expansion in caAkt Tg mice by decreased proliferation. CaAktTg mice deficient in cdk4 developed ␤-cell failure and diabetes. These experiments suggest that Akt induces ␤-cell proliferation in a cdk4-dependent manner by regulation of cyclin D1, cyclin D2, and p21 levels. These data also indicate that alteration in levels of these cell cycle components could affect the maintenance of ␤-cell mass in basal states and the adaptation of ␤-cells to pathological states resulting in diabetes. Diabetes 55:318 -325, 2006 T he defects that result in diabetes are diverse, but pancreatic ␤-cell failure is a major component of this disease (1,2). Pancreatic ␤-cell mass results from a dynamic balance of neogenesis, proliferation, cell size, and apoptosis (3). Recent data using cell lineage tracing experiments suggest that replication is a major component involved in maintenance of ␤-cell mass in adult animals (4). The molecular mechanisms involved in regulation of ␤-cell mass are unknown. Several lines of evidence suggest that activation of phosphoinositide 3-kinase (PI 3-kinase) signaling pathway plays critical roles in regulation of ␤-cell mass and function (5-10).One of the major targets of PI 3-kinase is the serinethreonine kinase Akt, the cellular homolog of the viral oncogene v-Akt (11). Akt is a convergent point of several growth signals induced by growth factors, insulin, and gut incretins. Transgenic mice overexpressing a constitutively active form of Akt in islet ␤-cells (caAkt Tg ) have provided further evidence for the role of this kinase in islet physiology (12,13). These mice exhibit striking increases in ␤-cell mass, proliferation, neogenesis, and cell size. Akt regulates cell cycle progression by alterations in levels and subcellular distribution of components of the cell cycle machinery (14). The molecules and the mechanisms involved in the regulation of cell cycle by growth signals mediated by Akt in pancreatic ␤-cells are ill defined.Insight into the mechanisms involved in the regulation of cell cycle in ␤-cells have been provided by experiments in cyclin-dependent kinase-4 (cdk4)-deficient mice. These mice exhibited growth retardation and diabetes du...

Osteoclasts express high levels of vacuolar H(+)-ATPase (V-ATPase) in their ruffled membranes, driving the secretion of H+ required for normal bone resorption. Previous reports have suggested that the B subunit of the osteoclast V-ATPase differs from those expressed in kidney and other tissues. In this study, B subunit isoform-specific antibodies and cDNA probes were used to examine which B subunit isoform is expressed in osteoclasts and osteoclast-like cells. Immunoblotting and RNA hybridization analysis were used to demonstrate that cells from an osteoclast-rich mouse bone marrow culture model express the B2 but not the B1 subunit isoform. Immunocytochemical staining of murine osteoclasts generated in vitro and of native rat osteoclasts in bone sections showed that the B2 but not the B1 isoform was expressed at high levels and was polarized to the ruffled membrane. Human marrow cultures and monocyte-derived macrophages, used as models for osteoclasts, also expressed the B2 but not the B1 subunit isoform. These results indicate that V-ATPases containing the B2 subunit isoform mediate osteoclast bone resorption.

Osteoclasts resorb bone through a cyclical process of attachment to matrix, polarization, retraction, and migration. Although this process requires major alterations in the organization of actin structures, little is known about roles that myosins play in osteoclast cytoskeletal dynamics. We performed immunolocalization of myosin II using antibodies against heavy chain isoforms IIA and IIB and found that osteoclasts expressed the isoforms in distinct subcellular locations. Myosin IIA was enriched in dynamic cytoskeletal compartments, including the sealing zones of polarized and unpolarized osteoclasts. In contrast, myosin IIB was generally absent from these regions and maintained a comparatively static distribution during different phases of the osteoclast activation cycle. Inhibition of myosin II in osteoclasts by treatment with 2,3-butanedione monoxime caused detachment of unpolarized, but not polarized, cells from the bone matrix. These results suggest that myosin IIA is critical to development of an activated osteoclast phenotype.

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