Combined Inhibition of DYRK1A, SMAD, and Trithorax Pathways Synergizes to Induce Robust Replication in Adult Human Beta Cells.

Small-molecule inhibitors of dual-specificity tyrosine-regulated kinase 1A (DYRK1A) induce human beta cells to proliferate, generating a labeling index of 1.5%-3%. Here, we demonstrate that combined pharmacologic inhibition of DYRK1A and transforming growth factor beta superfamily (TGFβSF)/SMAD signaling generates remarkable further synergistic increases in human beta cell proliferation (average labeling index, 5%-8%, and as high as 15%-18%), and increases in both mouse and human beta cell numbers.

Insights into beta cell regeneration for diabetes via integration of molecular landscapes in human insulinomas.

Although diabetes results in part from a deficiency of normal pancreatic beta cells, inducing human beta cells to regenerate is difficult. Reasoning that insulinomas hold the "genomic recipe" for beta cell expansion, we surveyed 38 human insulinomas to obtain insights into therapeutic pathways for beta cell regeneration. An integrative analysis of whole-exome and RNA-sequencing data was employed to extensively characterize the genomic and molecular landscape of insulinomas relative to normal beta cells.

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

β-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 β-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.

Augmented Stat5 Signaling Bypasses Multiple Impediments to Lactogen-Mediated Proliferation in Human β-Cells.

Pregnancy in rodents is associated with a two- to threefold increase in β-cell mass, which is attributable to large increases in β-cell proliferation, complimented by increases in β-cell size, survival, and function and mediated mainly by the lactogenic hormones prolactin (PRL) and placental lactogens. In humans, however, β-cell mass does not increase as dramatically during pregnancy, and PRL fails to activate proliferation in human islets in vitro. To determine why, we explored the human PRL-prolactin receptor (hPRLR)-Janus kinase 2 (JAK2)-signal transducer and activator of transcription 5 (STAT5)-cyclin-cdk signaling cascade in human β-cells.

Cytoplasmic-nuclear trafficking of G1/S cell cycle molecules and adult human β-cell replication: a revised model of human β-cell G1/S control.

Harnessing control of human β-cell proliferation has proven frustratingly difficult. Most G1/S control molecules, generally presumed to be nuclear proteins in the human β-cell, are in fact constrained to the cytoplasm. Here, we asked whether G1/S molecules might traffic into and out of the cytoplasmic compartment in association with activation of cell cycle progression.

Human pancreatic β-cell G1/S molecule cell cycle atlas.

Expansion of pancreatic β-cells is a key goal of diabetes research, yet induction of adult human β-cell replication has proven frustratingly difficult. In part, this reflects a lack of understanding of cell cycle control in the human β-cell. Here, we provide a comprehensive immunocytochemical "atlas" of G1/S control molecules in the human β-cell.

Overexpression of hepatocyte nuclear factor-4α initiates cell cycle entry, but is not sufficient to promote β-cell expansion in human islets.

The transcription factor HNF4α (hepatocyte nuclear factor-4α) is required for increased β-cell proliferation during metabolic stress in vivo. We hypothesized that HNF4α could induce proliferation of human β-cells. We employed adenoviral-mediated overexpression of an isoform of HNF4α (HNF4α8) alone, or in combination with cyclin-dependent kinase (Cdk)6 and Cyclin D3, in human islets.

ChREBP mediates glucose-stimulated pancreatic β-cell proliferation.

Glucose stimulates rodent and human β-cell replication, but the intracellular signaling mechanisms are poorly understood. Carbohydrate response element-binding protein (ChREBP) is a lipogenic glucose-sensing transcription factor with unknown functions in pancreatic β-cells. We tested the hypothesis that ChREBP is required for glucose-stimulated β-cell proliferation.

c-myc and skp2 coordinate p27 degradation, vascular smooth muscle proliferation, and neointima formation induced by the parathyroid hormone-related protein.

Parathyroid hormone-related protein (PTHrP) contains a classical bipartite nuclear localization signal. Nuclear PTHrP induces proliferation of arterial vascular smooth muscle cells (VSMC). In the arterial wall, PTHrP is markedly up-regulated in response to angioplasty and promotes arterial restenosis.

Regulated and reversible induction of adult human β-cell replication.

Induction of proliferation in adult human β-cells is challenging. It can be accomplished by introduction of cell cycle molecules such as cyclin-dependent kinase 6 (cdk6) and cyclin D1, but their continuous overexpression raises oncogenic concerns. We attempted to mimic normal, transient, perinatal human β-cell proliferation by delivering these molecules in a regulated and reversible manner.

A human islet cell culture system for high-throughput screening.

A small-molecule inducer of beta-cell proliferation in human islets represents a potential regeneration strategy for treating type 1 diabetes. However, the lack of suitable human beta cell lines makes such a discovery a challenge. Here, we adapted an islet cell culture system to high-throughput screening to identify such small molecules.

Activation of protein kinase C-ζ in pancreatic β-cells in vivo improves glucose tolerance and induces β-cell expansion via mTOR activation.

Objective: PKC-ζ activation is a key signaling event for growth factor-induced β-cell replication in vitro. However, the effect of direct PKC-ζ activation in the β-cell in vivo is unknown. In this study, we examined the effects of PKC-ζ activation in β-cell expansion and function in vivo in mice and the mechanisms associated with these effects.

cMyc is a principal upstream driver of beta-cell proliferation in rat insulinoma cell lines and is an effective mediator of human beta-cell replication.

Adult human β-cells replicate slowly. Also, despite the abundance of rodent β-cell lines, there are no human β-cell lines for diabetes research or therapy. Prior studies in four commonly studied rodent β-cell lines revealed that all four lines displayed an unusual, but strongly reproducible, cell cycle signature: an increase in seven G(1)/S molecules, i.

Parathyroid hormone-related protein enhances human ß-cell proliferation and function with associated induction of cyclin-dependent kinase 2 and cyclin E expression.

Objective: Inducing human β-cell growth while enhancing function is a major goal in the treatment of diabetes. Parathyroid hormone-related protein (PTHrP) enhances rodent β-cell growth and function through the parathyroid hormone-1 receptor (PTH1R). Based on this, we hypothesized that PTH1R is expressed in human β-cells and that PTHrP has the potential to enhance human β-cell proliferation and/or function.

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

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.

Novel proapoptotic effect of hepatocyte growth factor: synergy with palmitate to cause pancreatic {beta}-cell apoptosis.

Increasing evidence suggests that elevation of plasma fatty acids that often accompanies insulin resistance contributes to beta-cell insufficiency in obesity-related type 2 diabetes. Circulating levels of hepatocyte growth factor (HGF) are increased in humans with metabolic syndrome and obesity. HGF is known to protect beta-cells against streptozotocin and during islet engraftment.

Plasma membrane subdomain compartmentalization contributes to distinct mechanisms of ceramide action on insulin signaling.

Objective: Ceramide is now recognized as a negative regulator of insulin signaling by impairing protein kinase B (PKB)/Akt activation. In different cells, two distinct mechanisms have been proposed to mediate ceramide inhibition of PKB/Akt: one involving atypical protein kinase C zeta (PKCzeta) and the other the protein phosphatase-2 (PP2A). We hypothesized that ceramide action through PKCzeta or PP2A might depend on plasma membrane (PM) structural organization and especially on caveolin-enriched domain (CEM) abundance.

Survey of the human pancreatic beta-cell G1/S proteome reveals a potential therapeutic role for cdk-6 and cyclin D1 in enhancing human beta-cell replication and function in vivo.

Objectives: To comprehensively inventory the proteins that control the G1/S cell cycle checkpoint in the human islet and compare them with those in the murine islet, to determine whether these might therapeutically enhance human beta-cell replication, to determine whether human beta-cell replication can be demonstrated in an in vivo model, and to enhance human beta-cell function in vivo.

Research Design And Methods: Thirty-four G1/S regulatory proteins were examined in human islets. Effects of adenoviruses expressing cdk-6, cdk-4, and cyclin D1 on proliferation in human beta-cells were studied in both in vitro and in vivo models.

Mutant parathyroid hormone-related protein, devoid of the nuclear localization signal, markedly inhibits arterial smooth muscle cell cycle and neointima formation by coordinate up-regulation of p15Ink4b and p27kip1.

Arterial expression of PTH-related protein is markedly induced by angioplasty. PTH-related protein contains a nuclear localization signal (NLS). PTH-related protein mutants lacking the NLS (DeltaNLS-PTH-related protein) are potent inhibitors of arterial vascular smooth muscle cell (VSMC) proliferation in vitro.

Hepatocyte growth factor enhances engraftment and function of nonhuman primate islets.

Objective: Adenoviral delivery of hepatocyte growth factor (HGF) to rodent islets improves islet graft survival and function, markedly reducing the number of islets required to achieve glucose control. Here, we asked whether these prior observations in rodent models extend to nonhuman primate (NHP) islets.

Research Design And Methods: NHP islets were transduced with murine (Ad.

Lessons from the first comprehensive molecular characterization of cell cycle control in rodent insulinoma cell lines.

Objective: Rodent insulinoma cell lines may serve as a model for designing continuously replicating human beta-cell lines and provide clues as to the central cell cycle regulatory molecules in the beta-cell.

Research Design And Methods: We performed a comprehensive G1/S proteome analysis on the four most widely studied rodent insulinoma cell lines and defined their flow cytometric profiles and growth characteristics.

Results: 1) Despite their common T-antigen-derived origins, MIN6 and BTC3 cells display markedly different G1/S expression profiles; 2) despite their common radiation origins, RINm5F and INS1 cells display striking differences in cell cycle protein profiles; 3) phosphorylation of pRb is absent in INS1 and RINm5F cells; 4) cyclin D2 is absent in RINm5F and BTC3 cells and therefore apparently dispensable for their proliferation; 5) every cell cycle inhibitor is upregulated, presumably in a futile attempt to halt proliferation; 6) among the G1/S proteome members, seven are pro-proliferation molecules: cyclin-dependent kinase-1, -2, -4, and -6 and cyclins A, E, and D3; and 7) overexpression of the combination of these seven converts arrested proliferation rates in primary rat beta-cells to those in insulinoma cells.

Lactogens promote beta cell survival through JAK2/STAT5 activation and Bcl-XL upregulation.

One of the goals in the treatment for diabetes is to enhance pancreatic beta cell function, proliferation, and survival. This study explores the role of lactogenic hormones, prolactin (PRL) and placental lactogen (PL), in beta cell survival. We have previously shown that transgenic mice expressing mouse placental lactogen-1 (mPL1) in beta cells under the rat insulin II promoter (RIP) are resistant to the diabetogenic and cytotoxic effects of streptozotocin (STZ) in vivo.

Protein kinase C-zeta activation markedly enhances beta-cell proliferation: an essential role in growth factor mediated beta-cell mitogenesis.

Objective: Diabetes results from a deficiency of functional beta-cells. Previous studies have identified hepatocyte growth factor (HGF) and parathyroid hormone-related protein (PTHrP) as two potent beta-cell mitogens. The objective of this study is to determine 1) whether HGF and PTHrP have additive/synergistic effects on beta-cell growth and proliferation; 2) the signaling pathways through which these growth factors mediate beta-cell mitogenesis; and 3) whether activation of this/these signaling pathway(s) enhances human beta-cell replication.

Cellular mechanism through which parathyroid hormone-related protein induces proliferation in arterial smooth muscle cells: definition of an arterial smooth muscle PTHrP/p27kip1 pathway.

Parathyroid hormone-related protein (PTHrP) is present in vascular smooth muscle (VSM), is markedly upregulated in response to arterial injury, is essential for normal VSM proliferation, and also markedly accentuates neointima formation following rat carotid angioplasty. PTHrP contains a nuclear localization signal (NLS) through which it enters the nucleus and leads to marked increases in retinoblastoma protein (pRb) phosphorylation and cell cycle progression. Our goal was to define key cell cycle molecules upstream of pRb that mediate cell cycle acceleration induced by PTHrP.

Molecular control of cell cycle progression in the pancreatic beta-cell.

Type 1 and type 2 diabetes both result from inadequate production of insulin by the beta-cells of the pancreatic islet. Accordingly, strategies that lead to increased pancreatic beta-cell mass, as well as retained or enhanced function of islets, would be desirable for the treatment of diabetes. Although pancreatic beta-cells have long been viewed as terminally differentiated and irreversibly arrested, evidence now indicates that beta-cells can and do replicate, that this replication can be enhanced by a variety of maneuvers, and that beta-cell replication plays a quantitatively significant role in maintaining pancreatic beta-cell mass and function.