Novel contributions of Pdx-1 to pancreatic β-cell adaptation to insulin resistance

Type 2 diabetes results from pancreatic 13-cell failure in the setting of insulin resistance. Normal β-cell compensation for an increased insulin demand includes both enhanced insulin secretory capacity and an expansion of morphological β-cell mass due largely to changes in the balance between cell proliferation and apoptosis. Heterozygous mutations in the gene encoding the β-cell transcription factor Pdx1 are associated with type 2 diabetes in humans and reduced Pdx1 levels accompany β-cell dysfunction in several experimental models of the disease, suggesting that Pdx1 may regulate genes critical for the islet compensatory response. In the first part of my thesis, we describe the effect of Pdx1 haploinsufficiency on the β-cell response to obesity-associated insulin resistance in vivo by studying Pdx1+/- mice fed a high-fat diet (HFD). We demonstrate that HFD-fed Pdx1+/- mice develop diabetes due in part to decreased β-cell survival associated with increased endoplasmic reticulum (ER) stress, a feature of human type 2 diabetic islets. Furthermore, we show that Pdx1 lies upstream of several ER-related genes and directly regulates expression of the unfolded protein response transcription factor Atf4, uncovering a potential mechanism by which Pdx1 loss-of-function predisposes to β-cell failure in the setting of insulin resistance. Pdx1 can regulate a small subset of target genes as a heterodimer with the DNA-binding cofactor Pbx1 although little is known about the specific roles of this complex in the β-cell. In the second and third chapters of my thesis, we use both a genome-wide ChIP on chip approach and a hypothesis-driven strategy to identify the first direct targets of the Pdx1/Pbx1 heterodimer in the β-cell and consider their potential contribution to β-cell compensation. Therefore, altogether our results provide novel insight into the role of Pdx1 in β-cell adaptation to insulin resistance and provide the basis for future experiments to define the involvement of the Pdx1/Pbx1 heterodimer in this process

Novel contributions of Pdx-1 to pancreatic β-cell adaptation to insulin resistance

Type 2 diabetes results from pancreatic 13-cell failure in the setting of insulin resistance. Normal β-cell compensation for an increased insulin demand includes both enhanced insulin secretory capacity and an expansion of morphological β-cell mass due largely to changes in the balance between cell proliferation and apoptosis. Heterozygous mutations in the gene encoding the β-cell transcription factor Pdx1 are associated with type 2 diabetes in humans and reduced Pdx1 levels accompany β-cell dysfunction in several experimental models of the disease, suggesting that Pdx1 may regulate genes critical for the islet compensatory response. In the first part of my thesis, we describe the effect of Pdx1 haploinsufficiency on the β-cell response to obesity-associated insulin resistance in vivo by studying Pdx1+/- mice fed a high-fat diet (HFD). We demonstrate that HFD-fed Pdx1+/- mice develop diabetes due in part to decreased β-cell survival associated with increased endoplasmic reticulum (ER) stress, a feature of human type 2 diabetic islets. Furthermore, we show that Pdx1 lies upstream of several ER-related genes and directly regulates expression of the unfolded protein response transcription factor Atf4, uncovering a potential mechanism by which Pdx1 loss-of-function predisposes to β-cell failure in the setting of insulin resistance. Pdx1 can regulate a small subset of target genes as a heterodimer with the DNA-binding cofactor Pbx1 although little is known about the specific roles of this complex in the β-cell. In the second and third chapters of my thesis, we use both a genome-wide ChIP on chip approach and a hypothesis-driven strategy to identify the first direct targets of the Pdx1/Pbx1 heterodimer in the β-cell and consider their potential contribution to β-cell compensation. Therefore, altogether our results provide novel insight into the role of Pdx1 in β-cell adaptation to insulin resistance and provide the basis for future experiments to define the involvement of the Pdx1/Pbx1 heterodimer in this process

Minireview: Meeting the Demand for Insulin: Molecular Mechanisms of Adaptive Postnatal ß-Cell Mass Expansion

Type 2 diabetes results from pancreatic ß-cell failure in the setting of insulin resistance. This model of disease progression has received recent support from the results of genome-wide association studies that identify genes potentially regulating ß-cell growth and function as type 2 diabetes susceptibility loci. Normal ß-cell compensation for an increased insulin demand includes both enhanced insulin-secretory capacity and an expansion of morphological ß-cell mass, due largely to changes in the balance between ß-cell proliferation and apoptosis. Recent years have brought significant progress in the understanding of both extrinsic signals stimulating ß-cell growth as well as mediators intrinsic to the ß-cell that regulate the compensatory response. Here, we review the current knowledge of mechanisms underlying adaptive expansion of ß-cell mass, focusing on lessons learned from experimental models of physiologically occurring insulin-resistant states including diet-induced obesity and pregnancy, and highlighting the potential importance of interorgan cross talk. The identification of critical mediators of islet compensation may direct the development of future therapeutic strategies to enhance the response of ß-cells to insulin resistance

Endophthalmitis following intravitreal injection of anti-VEGF agents: long-term outcomes and the identification of unusual micro-organisms

BackgroundWhile the development of targeted molecular therapy to inhibit vascular endothelial growth factor (VEGF) has revolutionized the treatment and visual prognosis of highly prevalent retinal diseases such as diabetic retinopathy and age-related macular degeneration, each intravitreal injection of these agents carries a small risk of endophthalmitis which can be visually devastating. In the absence of specific guidelines, current management of post-injection endophthalmitis is typically extrapolated from data regarding endophthalmitis occurring after cataract surgery despite potential differences in pathogenic organisms and clinical course. Here, we assess the contribution of intravitreal injections of anti-VEGF agents to all cases of endophthalmitis at our tertiary care referral center and characterize the clinical outcomes and microbial pathogens associated with post-injection endophthalmitis in order to inform management of this serious iatrogenic condition.ResultsDuring the 7-year study period analyzed, 199 cases of endophthalmitis were identified using billing records. Of these, the most common etiology was post-surgical, accounting for 62 cases (31.2 %), with bleb-associated, endogenous, and corneal ulcer-related infections representing the next most frequent causes, comprising 15.6 % (31/199), 13.1 % (26/199), and 13.6 % (27/199) of all cases, respectively. Intravitreal injections of anti-VEGF agents represented 8.5 % of endophthalmitis (17/199 cases). Intraocular cultures yielded positive results in 75 % of post-injection cases, with the majority associated with coagulase-negative Staphylococcus. Consistent with prior literature, a case of Strep viridans displayed more rapid onset and progression. We also report the first association of Enterobacter cloacae and Lactococcus garvieae with post-injection endophthalmitis. While all but one patient were treated with initial vitreous tap and intravitreal injection of antibiotics, both patients with these rare organisms exhibited persistent vitritis requiring subsequent vitrectomy. Long-term outcomes of post-injection endophthalmitis indicated visual recovery to baseline levels, even with resumption of anti-VEGF agents following resolution of the acute infection.ConclusionsAcute endophthalmitis following intravitreal injections of anti-VEGF agents is an uncommon but potentially devastating complication which may be managed effectively with vitreous tap and injection of intravitreal antibiotics. However, persistent vitritis requiring subsequent vitrectomy should raise suspicion for unusual pathogens

Human Embryonic Stem Cell-Derived Retinal Pigment Epithelial Cell Transplantation for Retinal Degeneration

This chapter summarizes the design and outcomes of the first clinical trials of human embryonic stem cell (hESC)-derived retinal pigment epithelial (RPE) cell transplantation therapy for retinal regeneration. The MA09-(hESC) line was derived from the blastomere stage of a donated embryo and expanded on mitotically inactivated mouse embryonic fibroblasts according to the Good Manufacturing Practices. The MA09-hRPE cells were then tested for safety and terminal differentiation into mature RPE cells by gene expression analysis, karyotyping, phagocytosis assay, and differentiation and purity evaluation by way of morphology, quantitative polymerase chain reaction, and quantitative immune staining for RPE and hESC markers. Two phase I/II open-label, multicenter, prospective clinical trials investigating the safety of subretinal injection of hESC-derived RPE cell suspension in patients with end-stage atrophic age-related macular degeneration (AMD) and Stargardt macular dystrophy (SMD) were performed. The visual outcomes were encouraging with some patients gaining more than ten letters in both groups; however, these results must be tempered by the lack of a control group, poor initial visual acuity, short follow-up, and limited number of patients. Thirteen of eighteen patients (72%) developed areas of increased subretinal pigmentation, some of which appeared to correlate to hyper-reflective bands on optical coherence tomography. The transplanted cells showed no evidence of tumor formation, adverse preretinal RPE cell engraftment, or clinically apparent rejection. Aside from a case of acute postoperative endophthalmitis, there were no issues with the surgical procedure itself. These promising results suggest that hESC-derived RPE cells could represent a novel treatment paradigm for retinal degenerations hallmarked by tissue loss or dysfunction

Profile of Histone Lysine Methylation across Transcribed Mammalian Chromatin

Complex patterns of histone lysine methylation encode distinct functions within chromatin. We previously reported that trimethylation of lysine 9 of histone H3 (H3K9) occurs at both silent heterochromatin and at the transcribed regions of active mammalian genes, suggesting that the extent of histone lysine methylation involved in mammalian gene activation is not completely defined. To identify additional sites of histone methylation that respond to mammalian gene activity, we describe here a comparative assessment of all six known positions of histone lysine methylation and relate them to gene transcription. Using several model loci, we observed high trimethylation of H3K4, H3K9, H3K36, and H3K79 in the transcribed region, consistent with previous findings. We identify H4K20 monomethylation, a modification previously linked with repression, as a mark of transcription elongation in mammalian cells. In contrast, H3K27 monomethylation, a modification enriched at pericentromeric heterochromatin, was observed broadly distributed throughout all euchromatic sites analyzed, with selective depletion in the vicinity of the transcription start sites at active genes. Together, these results underscore that similar to other described methyl-lysine modifications, H4K20 and H3K27 monomethylation are versatile and dynamic with respect to gene activity, suggesting the existence of novel site-specific methyltransferases and demethylases coupled to the transcription cycle

Pcif1 modulates Pdx1 protein stability and pancreatic β cell function and survival in mice

The homeodomain transcription factor pancreatic duodenal homeobox 1 (Pdx1) is a major mediator of insulin transcription and a key regulator of the β cell phenotype. Heterozygous mutations in PDX1 are associated with the development of diabetes in humans. Understanding how Pdx1 expression levels are controlled is therefore of intense interest in the study and treatment of diabetes. Pdx1 C terminus–interacting factor-1 (Pcif1, also known as SPOP) is a nuclear protein that inhibits Pdx1 transactivation. Here, we show that Pcif1 targets Pdx1 for ubiquitination and proteasomal degradation. Silencing of Pcif1 increased Pdx1 protein levels in cultured mouse β cells, and Pcif1 heterozygosity normalized Pdx1 protein levels in Pdx1+/– mouse islets, thereby increasing expression of key Pdx1 transcriptional targets. Remarkably, Pcif1 heterozygosity improved glucose homeostasis and β cell function and normalized β cell mass in Pdx1+/– mice by modulating β cell survival. These findings indicate that in adult mouse β cells, Pcif1 limits Pdx1 protein accumulation and thus the expression of insulin and other gene targets important in the maintenance of β cell mass and function. They also provide evidence that targeting the turnover of a pancreatic transcription factor in vivo can improve glucose homeostasis

Pcif1 modulates Pdx1 protein stability and pancreatic beta cell function and survival in mice

The homeodomain transcription factor pancreatic duodenal homeobox 1 (Pdx1) is a major mediator of insulin transcription and a key regulator of the β cell phenotype. Heterozygous mutations in PDX1 are associated with the development of diabetes in humans. Understanding how Pdx1 expression levels are controlled is therefore of intense interest in the study and treatment of diabetes. Pdx1 C terminus-interacting factor-1 (Pcif1, also known as SPOP) is a nuclear protein that inhibits Pdx1 transactivation. Here, we show that Pcif1 targets Pdx1 for ubiquitination and proteasomal degradation. Silencing of Pcif1 increased Pdx1 protein levels in cultured mouse β cells, and Pcif1 heterozygosity normalized Pdx1 protein levels in Pdx1 +/-mouse islets, thereby increasing expression of key Pdx1 transcriptional targets. Remarkably, Pcif1 heterozygosity improved glucose homeostasis and β cell function and normalized β cell mass in Pdx1 +/-mice by modulating β cell survival. These findings indicate that in adult mouse β cells, Pcif1 limits Pdx1 protein accumulation and thus the expression of insulin and other gene targets important in the maintenance of β cell mass and function. They also provide evidence that targeting the turnover of a pancreatic transcription factor in vivo can improve glucose homeostasis