PANDER (PANcreatic DERived factor, FAM3B), a newly discovered secreted cytokine, is specifically expressed at high levels in the islets of Langerhans of the endocrine pancreas. To evaluate the role of PANDER in ␤-cell function, we investigated the effects of PANDER on rat, mouse, and human pancreatic islets; the ␤-TC3 cell line; and the ␣-TC cell line. PANDER protein was present in ␣-and ␤-cells of pancreatic islets, insulinsecreting ␤-TC3 cells, and glucagon-secreting ␣-TC cells. PANDER induced islet cell death in rat and human islets. Culture of ␤-TC3 cells with recombinant PANDER had a dose-dependent inhibitory effect on cell viability. This effect was also time-dependent. PANDER caused apoptosis of ␤-cells as assessed by electron microscopy, annexin V fluorescent staining, and flow-cytometric terminal deoxynucleotidyl transferase-mediated dUTP nickend labeling assay. PANDER did not affect cytosolic Ca 2؉ levels or nitric oxide levels. However, PANDER activated caspase-3. Hence, PANDER may have a role in the process of pancreatic ␤-cell apoptosis.

Pancreatic-derived factor (PANDER) is an islet-specific cytokine present in both pancreatic ␣-and ␤-cells, which, in vitro, induces ␤-cell apoptosis of primary islet and cell lines. In this study, we investigated whether PANDER is secreted by pancreatic ␣-and ␤-cells and whether PANDER secretion is regulated by glucose and other insulin secretagogues. In mouse-derived insulin-secreting ␤-TC3 cells, PANDER secretion in the presence of stimulatory concentrations of glucose was 2.8 ؎ 0.4-fold higher (P < 0.05) than without glucose. Insulin secretion was similarly increased by glucose in the same cells. The total concentration of secreted PANDER in the medium was ϳ6 -10 ng/ml [IFN]-␥) play vital roles in ␤-cell dysfunction and death and in the development of type 1 diabetes (2-6). Recently, it was shown that glucose causes islet ␤-cells to produce IL-1␤, while the released IL-1␤ has a deleterious effect on human pancreatic islets (7,8). These studies suggest that production and release of cytokines from pancreatic islet cells are involved in ␤-cell dysfunction and death in hyperglycemia. To date, although several cytokines have been shown to be involved in ␤-cell dysfunction and death, the precise mechanisms of type 1 diabetes are still incompletely understood, suggesting that other potential factors may be involved.Pancreatic-derived factor (PANDER), also known as FAM3B (9,10), is one of four members of a new cytokine family recently identified using the algorithm ostensible recognition of folds (11) while searching for novel cytokines based on their predicted secondary structure. The rationale for this approach is that the secondary structure of cytokines is highly conserved through evolution. Many cytokines, such as IL

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PANDER (pancreatic derived factor, FAM3B) is a novel cytokine, present in insulin secretory granules, that induces apoptosis of alpha and beta cells of mouse, rat, and human islets in a dose- and time-dependent manner, and may be implicated in diabetes. PANDER has the predicted secondary structure of 4 alpha-helical bundles with an up-up-down-down topology, and two disulfide bonds. Eleven mutated PANDERs were constructed and expressed in beta-TC3 cells to identify the essential region of PANDER involved in beta-cell death. Beta-cell function was assessed by assays of cell viability and insulin secretion. Based on quantitative real-time RT-PCR all mutant PANDERs had similar mRNA expression levels in beta-TC3 cells. Immunoblotting showed that ten of eleven mutant PANDER proteins were synthesized and detected in beta-TC3 cells. A mutant PANDER with no signal peptide, however, was not expressed. Truncation of helix D alone caused a 40-50% decrease in PANDER's activity, while truncation of both helices C and D resulted in a 75% loss of activity. In contrast, truncation of the N-terminus of PANDER (helix A, the loop between helices A and B, and the first two cysteines) had no effect on PANDER-induced beta-cell death. The third and fourth cysteines of PANDER, C91 and C229, were shown to form one disulfide bond and be functionally important. Finally, the region between Cys91 and Phe152 constitutes the active part of PANDER, based on the demonstration that mutants with truncation of helix B or C caused decreased beta-cell death and did not inhibit insulin secretion, as compared to wild-type PANDER. Hence, helices B and C and the second disulfide bond of PANDER are essential for PANDER-induced beta-cell death.

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Efficient production of cloned embryos and live offspring is dependent on the ability of the nuclear-donor cell to be reprogrammed to direct normal conceptus development. Results of comparative studies indicate that embryonic and fetal cells are more successful candidates for nuclear transfer (NT) than terminally differentiated cells. Comparison of donor-cell efficiency is difficult to interpret across laboratories and from donor animals of varying genetic backgrounds and tissues of origin. This study was undertaken to determine the effect of the stage of differentiation of adult somatic donor cells derived from one-cell lineage of an individual donor animal. The follicular cell lineage including preantral follicle (PAFC), cumulus (CC), granulosa (GC), and luteal cells (LC) was chosen as several cell types in the lineage have previously proven successful for NT. Donor cell cultures were established from a 3-year-old Holstein cow. Embryos were reconstructed using confluent, early passage cultures. For each replicate, embryos were produced from two donor cell types in randomized block design (14 trials). Viable embryos were transferred to recipient females after 7 days of in vitro culture. Pregnancy and fetal viability were monitored weekly by ultrasonography from Days 30-100 of gestation and by rectal palpation thereafter. Embryos reconstructed from PAFC were less likely to develop to the blastocyst stage on Day 7 than embryos derived from CC or LC (Table 1, P < 0.05). Pregnancy rates at Day 30 were similar across donor cell types. A greater proportion of PAFC embryos were viable at Day 60 of gestation than embryos derived from CC and GC (P < 0.05). To date, normal cloned calves have been delivered at term from CC and GC, and two pregnancies (n = 3 fetuses) are ongoing from PAFC. The fetus cloned from LC, the terminally differentiated cell type in the lineage, was spontaneously aborted at day 211 with congenital abnormalities. Results from comparative studies of development of mouse embryos cloned from embryonic stem (ES) cells v. somatic donor cells indicate that ES-derived clones are less efficient in blastocyst formation, but survival to term is greater (Humpherys D et al., 2002 PNAS 99, 12 889-12 894). Likewise, our results in cattle suggest that PAFC, the least differentiated cells in the lineage, result in fewer cloned blastocysts, but blastocysts that do develop are more likely to progress through implantation and into later stages of pregnancy. Although high blastocyst rates can be achieved in somatic cell nuclear transfer, abortions and developmental abnormalities still hamper advancement. Reliable and practical methods to evaluate early embryonic development and differentiation are required to understand and overcome the problem. Our aim was to establish an in vitro culture system for monitoring posthatching development (PHD). Slaughterhouse-derived bovine oocytes were matured in vitro, fertilized (Day 0) and cultured (Holm et al., 1999, Theriogenology, 52, 683-700). On Day 8, degenerated embryos were ...