Definition of a Skp2-c-Myc Pathway to Expand Human Beta-cells.

Type 2 diabetes (T2D) is characterized by insulin resistance and reduced functional ß-cell mass. Developmental differences, failure of adaptive expansion and loss of ß-cells via ß-cell death or de-differentiation have emerged as the possible causes of this reduced ß-cell mass. We hypothesized that the proliferative response to mitogens of human ß-cells from T2D donors is reduced, and that this might contribute to the development and progression of T2D. Here, we demonstrate that the proliferative response of human ß-cells from T2D donors in response to cdk6 and cyclin D3 is indeed dramatically impaired. We show that this is accompanied by increased nuclear abundance of the cell cycle inhibitor, p27(kip1). Increasing nuclear abundance of p27(kip1) by adenoviral delivery decreases the proliferative response of ß-cells from non-diabetic donors, mimicking T2D ß-cells. However, while both p27(kip1) gene silencing and downregulation by Skp2 overexpression increased similarly the proliferative response of human ß-cells, only Skp2 was capable of inducing a significant human ß-cell expansion. Skp2 was also able to double the proliferative response of T2D ß-cells. These studies define c-Myc as a central Skp2 target for the induction of cell cycle entry, expansion and regeneration of human T2D ß-cells.

Episodes of clinical mastitis and its relationship with duration of treatment and seasonality in crossbred cows maintained in organized dairy farm.

AIM: Present study aimed to evaluate the different episodes of clinical mastitis (CM) and influence of duration of treatment and seasonality on the occurrence of different episodes of CM in crossbred cows. MATERIALS AND METHODS: A total of 1194 lactation data of crossbred CM cows were collected from mastitis treatment record from 2002 to 2012. Data of CM cows were classified into types of episodes (pattern of repeated or multiple episodes occurrence) and number of episodes (magnitude of multiple cases). Types of episodes were divided as single (clinical cure by a single episode of treatment), relapse (retreatment of the same cow within 21 days), recurrence (new CM at least 21 days after treatment), and both (relapse and recurrence). The season was classified as winter (December to March), summer (April to June), rainy (July to September), and autumn (October to November). The difference between incidences of different types of CM episodes and the association between number or type of CM episodes with duration of treatment and seasons of CM occurrence were analyzed by Chi-square test. RESULTS: Among 1194 animals suffered with CM, 53, 16, and 18% had the single episode, relapse, and recurrence, respectively; while 13% suffered by both relapse and recurrence. We estimated the duration of treatment and found 80% of the cows treated 1-8 days, in which 65% treated for 1-4 days, while 35% cows were treated for 5-8 days. Further, 12% cows treated for 9-15 days and 7.5% cows treated >15 days. The relationship between duration of treatment and different episodes of CM revealed that 1-8 days treated cows were mostly cured by the single episode with less relapse and recurrence. In contrast, the incidences of recurrence and relapse episodes were higher in cows treated for more than 9 days. The highest incidence of relapse was noticed in winter (36%) than other seasons (10-28%), while the recurrence was less during autumn (9%) compared to other seasons (20-40%). CONCLUSION: Cows those suffered by both relapse and recurrence were more susceptible to CM, and they need to be culled from farm to control the transmission of infections. Although the influence of seasonality was difficult to understand, the higher magnitude of relapse and recurrence during winter suggested the adverse effects of cold stress on treatment outcome.

Early and Late G1/S Cyclins and Cdks Act Complementarily to Enhance Authentic Human ß-Cell Proliferation and Expansion.

ß-Cell regeneration is a key goal of diabetes research. Progression through the cell cycle is associated with retinoblastoma protein (pRb) inactivation via sequential phosphorylation by the "early" cyclins and cyclin-dependent kinases (cdks) (d-cyclins cdk4/6) and the "late" cyclins and cdks (cyclin A/E and cdk1/2). In ß-cells, activation of either early or late G1/S cyclins and/or cdks is an efficient approach to induce cycle entry, but it is unknown whether the combined expression of early and late cyclins and cdks might have synergistic or additive effects. Thus, we explored whether a combination of both early and late cyclins and cdks might more effectively drive human ß-cell cell cycle entry than either group alone. We also sought to determine whether authentic replication with the expansion of adult human ß-cells could be demonstrated. Late cyclins and cdks do not traffic in response to the induction of replication by early cyclins and cdks in human ß-cells but are capable of nuclear translocation when overexpressed. Early plus late cyclins and cdks, acting via pRb phosphorylation on distinct residues, complementarily induce greater proliferation in human ß-cells than either group alone. Importantly, the combination of early and late cyclins and cdks clearly increased human ß-cell numbers in vitro. These findings provide additional insight into human ß-cell expansion. They also provide a novel tool for assessing ß-cell expansion in vitro.

RNAi screening in primary human hepatocytes of genes implicated in genome-wide association studies for roles in type 2 diabetes identifies roles for CAMK1D and CDKAL1, among others, in hepatic glucose regulation.

Genome-wide association (GWA) studies have described a large number of new candidate genes that contribute to of Type 2 Diabetes (T2D). In some cases, small clusters of genes are implicated, rather than a single gene, and in all cases, the genetic contribution is not defined through the effects on a specific organ, such as the pancreas or liver. There is a significant need to develop and use human cell-based models to examine the effects these genes may have on glucose regulation. We describe the development of a primary human hepatocyte model that adjusts glucose disposition according to hormonal signals. This model was used to determine whether candidate genes identified in GWA studies regulate hepatic glucose disposition through siRNAs corresponding to the list of identified genes. We find that several genes affect the storage of glucose as glycogen (glycolytic response) and/or affect the utilization of pyruvate, the critical step in gluconeogenesis. Of the genes that affect both of these processes, CAMK1D, TSPAN8 and KIF11 affect the localization of a mediator of both gluconeogenesis and glycolysis regulation, CRTC2, to the nucleus in response to glucagon. In addition, the gene CDKAL1 was observed to affect glycogen storage, and molecular experiments using mutant forms of CDK5, a putative target of CDKAL1, in HepG2 cells show that this is mediated by coordinate regulation of CDK5 and PKA on MEK, which ultimately regulates the phosphorylation of ribosomal protein S6, a critical step in the insulin signaling pathway.