Human and murine diabetes is characterized by pancreatic β-cell dedifferentiation, a process in which β-cells lose expression of markers of maturity and gain those of endocrine progenitors. Failing β-cells inappropriately metabolize lipids over carbohydrates and exhibit impaired mitochondrial oxidative phosphorylation. Therefore, pathways involved in mitochondrial fuel selection and catabolism may represent potential targets for the prevention or reversal of dedifferentiation. In chapter I of this dissertation, we isolated and functionally characterized failing β-cells from various experimental models of diabetes. We found a striking enrichment in the expression of aldehyde dehydrogenase 1 isoform A3 (Aldh1a3) as β-cells become dedifferentiated. Flow-sorted Aldh1a3-expressing (ALDH+) islet cells demonstrate impaired glucose-induced insulin secretion, are depleted of Foxo1 and MafA, and include a Neurogenin3-positive subset. RNA sequencing analysis demonstrated that ALDH+ cells are characterized by: (i) impaired oxidative phosphorylation and mitochondrial complex I, IV, and V; (ii) activated RICTOR; and (iii) progenitor cell markers. We propose that impaired mitochondrial function marks the progression from metabolic inflexibility to dedifferentiation in the natural history of β-cell failure. In chapter II of this dissertation, we report that cytochrome b5 reductase 3 (Cyb5r3) is a FoxO1-regulated mitochondrial oxidoreductase critical to β cell function. Expression of Cyb5r3 is greatly decreased in multiple murine models of diabetes, and in vitro Cyb5r3 knockdown leads to increased ROS generation and impairment of respiration, mitochondrial function, glucose-stimulated insulin secretion, and calcium mobilization. In vivo, mice with β-cell-specific ablation of Cyb5r3 (B-Cyb5r3) display impaired glucose tolerance with decreased insulin secretion, and their islets have significantly lower basal respiration and glucose-stimulated insulin secretion. B-Cyb5r3 β-cells lose expression of Glut2, MafA, and Pdx1 expression despite a compensatory increase in FoxO1 expression. Our data suggest that Cyb5r3 is a critical mediator of FoxO1’s protective response in β-cells, and that loss of Cyb5r3 expression is an early event in β-cell failure.

Thesis (Ph. D.)--School of Chemical Engineering, Georgia Institute of Technology, 2004. Directed by Athanassios Sambanis. / Includes bibliographical references (leaves 125-135).

Diabetes is a chronic disorder of the pancreas, where a decline in the insulin-producing β-cell population disrupts metabolic homeostasis. Pancreatic transplantation has shown to be effective in circumventing the problem of β-cell insufficiency. However, availability of donor islets remains an obstacle. Although progressive differentiation of embryonic stem cells (ESCs) to pancreatic β-cells is a solution, current protocols are wrought with inefficiencies. It is obvious that to realize ESC differentiation for therapy many steps need to be optimized, and this study describes improvement of Pdx1+pancreatic progenitor derivation, a critical determinant of pancreatic fate. The compounds melatonin and sPDZD2 have been suggested to act through the Protein Kinase A (PKA) pathway to exert transcriptional effects, and in particular sPDZD2 stimulates the expression of pancreatic genes in INS-1E rat pancreatic cells. This led to the hypothesis that the PKA-targeting characteristics of said molecules could be exploited for pancreatic specification through post-translational activation ofPdx1. hESCs were first induced to form definitive endoderm before treatment with melatonin and sPDZD2. Pdx1 expression induced by these molecules was then compared with levels triggered by known pancreatic progenitor inducer Indolactam V (ILV). A secondary objective of this study was to assess the endoderm induction potential of small molecules in hESCs, which claim to be potentially useful in differentiation. In this research, I show that small molecules are noticeably more challenging to use in the hESC context. Between the TGF-β pathwayactivatorsIDE-1 and 2, the latter is more potent at inducing endoderm formation, though it does not surpass the capabilities of Stauprimide, a molecule originally thought to only serve a priming purpose in mESCs.IDE-2 and Stauprimide consistently perform better than Activin A, the near universal factor for endoderm induction. Possible synergy between IDE-2 and Stauprimide was explored, but their combination appears detrimental to Sox17expression. Subsequent pancreatic differentiation was also inefficient, and my results affirm the immaturity of chemically-induced endoderm by contrasting with mainstream means of endoderm induction; levels of endoderm marker expression between the two methods are millions of folds apart. This work exposes the risks of using small molecules, and they necessitate proper characterization before being adopted for differentiation. Most favorably, both sPDZD2 and melatonin were able to trigger Pdx1 expression in STEMDiffTm derived definitive endoderm; 10 and 30folds respectively, comparable to the known Pdx1 inducer ILV (25 folds). I also reveal concentration-mediated differentiation and proliferative purposes of ILV and sPDZD2, which are highly reminiscent of the signaling mechanisms involved during pancreatic development. Preliminary quantification of Pdx1+ cells suggest that high concentrations of ILV and sPDZD2 favor self-renewal of Pdx1+ progenitors, whilst lower doses elevate Pdx1 expression. Demonstration of Pdx1 at both gene and protein expression levels was encouraging, but it remains uncertain if melatonin and sPDZD2 manipulate PKA signaling to exert Pdx1 promoting effects. My work supports the use of melatonin as a candidate for pancreatic differentiation, and suggests involvement of sPDZD2 in deriving and expanding progenitors during pancreatic organogenesis. / published_or_final_version / Biochemistry / Master / Master of Philosophy

published_or_final_version / Biochemistry / Doctoral / Doctor of Philosophy

Thesis (Ph.D.)--Aberdeen University, 2008. / Title from web page (viewed on July 14, 2009). Includes bibliographical references.

Dietary antioxidant curcumin derived from turmeric has been suggested to decrease the risk of many chronic diseases. Much of the existing data for curcumin stem from experiments performed at supra-physiological concentrations (μM-mM) that are impossible to attain through oral ingestion. It was therefore hypothesized that curcumin at low plasma achievable concentration, though itself not acting as a direct antioxidant might up-regulate the intracellular antioxidants and thus helping combat oxidative stress and protect β-islet cells. The results indicated that Curcumin, DMC and BDMC were able to scavenge hydroxyl radicals, but showed little scavenging ability against superoxide and nitric oxide radicals. Nanomolar concentrations of curcuminoids easily prevented the deleterious effects of H₂O₂ in pancreatic <i>β</i>-islet RINm5F cells. Non of the curcuminoids showed a detrimental effect on insulin secretion, but the model did not allow assessment of any potential positive effect on insulin secretion. The findings confirmed that nanomolar concentrations of curcumin offered protection in pancreatic <i>β</i>-islet cells against H₂O₂-indicated damage by modulating the proportion of oxidised GSH (GSSG): reduced GSH in the favour of GSH and the increasing the activity of SOD. This increase in GSH and SOD levels was, at least in part, on account of an increase in GR, SOD-1 and SOD-2 gene expression. The intracellular mechanism driving this modulation of antioxidant gene was, by virtue of blocking the H₂O₂ induced NF-κB activation.

No description available.

Indiana University-Purdue University Indianapolis (IUPUI) / Pancreatic insulin-secreting beta cell failure is central to the development of diabetes. Therapeutic applications targeted at understanding and manipulating beta cell destruction mechanisms should enhance the preservation of functional beta cell mass and prevent diabetes. To this end, we have demonstrated that diabetogenic assaults (e.g., endoplasmic reticulum stress, glucolipotoxicity, and pro-inflammatory cytokines) attenuate the activation of beta cell pro-survival signaling pathways via a stress-inducible molecule called Mitogen-inducible gene 6 (Mig6). We discovered that the overabundance of Mig6 exacerbates stress-induced beta cell apoptosis and inhibits insulin secretion. Conversely, the deficiency of Mig6 partially protected beta cells from DNA damage-induced cell death. Further, we established that Mig6 haploinsufficient mice retained islet integrity and function and exhibited greater beta cell mass recovery following treatment with multiple low doses of the beta cell toxin streptozotocin. These data suggest that Mig6 may be a therapeutic target for beta cell preservation in diabetes.

Embryonic stem (ES) cells were used as a model system to understand the signalling events in pancreas development. ES cells were differentiated through known precursor stages towards the tissue of interest in order to recapitulate development in vitro. Thus, protocols directing differentiation of mouse ES cells towards definitive endoderm and pancreatic β-cells were developed. A combination of activin A and bone morphogenic protein 4 resulted in a population of enriched cells expressing genetic markers of definitive endoderm. In vitro differentiation of ES cells into functional pancreatic β-cells has only been partially successful, as it results in cells that are not fully differentiated or functional. This might be due to a lack of cues emanating from surrounding cells present in the developing pancreas. Conditioned media from the mouse MIN6 β-cell line were used on the basis that differentiated β- cells might send out signals affecting the differentiation of the surrounding islet cells. Mouse ES cells were enriched in definitive endoderm and then treated with MIN6 conditioned medium. Gene expression of the β-cell markers Insulin1, Insulin2, and Glucose transporter 2 was significantly increased relative to the untreated control group after 10 days of treatment with conditioned medium. This result was specific for conditioned medium from MIN6 cells as conditioned medium from a kidney-, a neuronal-, and an exocrine pancreatic cell line had no effect. In order to characterise the secreted factor(s) the conditioned medium was subjected to protein precipitation. The pancreatic differentiation factor was present in a protein fraction, suggesting that the factor(s) was proteinaceous. The protein in question was neither proinsulin nor insulin. This knowledge will support the efficient generation of insulin-secreting cells for diabetes therapy.

Embryonic stem (ES) cells represent a possible source of islet tissue for the treatment of diabetes.  Achieving this goal will require a detailed understanding of how the transcription factor cascade initiated by the homeodomain transcription factor Pdx1 culminates in pancreatic beta-cell development.  Here we describe a genetic approach that enables fine control of Pdx1 transcriptional activity during endoderm differentiation of mouse ES cell.  By activating an exogenous Pdx1VP16 protein in populations of cells enriched in definitive endoderm we show a distinct lineage-dependent requirement for this transcription factor’s activity.  mimicking the natural biphasic pattern of Pdx1 expression was necessary to induce an endocrine pancreas-like cell phenotype, in which 30% of the cells were beta-cell-like.  Cell markers consistent with the different beta-cell differentiation stages appeared in a sequential order following the natural pattern of pancreatic development.  Furthermore, the differential beta-like cells secreted C-peptide (insulin) in response to KC1 and IBMX, suggesting that following a natural path of development in vitro represents the best approach to generate functional pancreatic cells.  Together these results reveal for the first time a significant effect of the timed expression of Pdx1 on the non-beta cells in the developing endocrine pancreas.  Collectively, we show that this method of <i>in vitro</i> differentiation provides a template for inducing and studying ES cell differentiation into insulin-secreting cells.