Anthony T. Cheung

An in vivo model for elucidation of the mechanism of tumor necrosis factor-alpha (TNF-alpha)-induced insulin resistance: evidence for differential regulation of insulin signaling by TNF-alpha.

Tumor necrosis factor-α (TNF-α) has been shown to induce insulin resistance in cultured cells as well as in animal models. The aim of this study was to map the in vivo mechanism whereby TNF-α contributes to the pathogenesis of impaired insulin signaling, using obese and lean Zucker rats in which TNF-α activity was inhibited through adenovirus-mediated gene transfer. We employed a replication-incompetent adenovirus-5 (Ad5) vector to endogenously express a TNF inhibitor (TNFi) gene, which encodes a chimeric protein consisting of the extracellular domain of the human 55-kDa TNF receptor joined to a mouse IgG heavy chain. Control animals consisted of rats infected with the same titer of adenovirus carrying the lac-z complementary DNA, encoding for β-galactosidase. There was a significant reduction in plasma insulin and free fatty acid levels in TNFi obese rats 2 days following Ad5 administration. The peripheral insulin sensitivity index was 50% greater, whereas hepatic glucose output was completely suppressed...

Improving function and survival of pancreatic islets by endogenous production of glucagon-like peptide 1 (GLP-1)

Glucagon-like peptide 1 (GLP-1) is a hormone that has received significant attention as a therapy for diabetes because of its ability to stimulate insulin biosynthesis and release and to promote growth and survival of insulin-producing β cells. While GLP-1 is produced from the proglucagon precursor by means of prohormone convertase (PC) 1/3 activity in enteroendocrine L cells, the same precursor is differentially processed by PC2 in pancreatic islet α cells to release glucagon, leaving GLP-1 trapped within a larger fragment with no known function. We hypothesized that we could induce GLP-1 production directly within pancreatic islets by means of delivery of PC1/3 and, further, that this intervention would improve the viability and function of islets. Here, we show that adenovirus-mediated expression of PC1/3 in α cells increases islet GLP-1 secretion, resulting in improved glucose-stimulated insulin secretion and enhanced survival in response to cytokine treatment. PC1/3 expression in α cells also improved performance after islet transplantation in a mouse model of type 1 diabetes, possibly by enhancing nuclear Pdx1 and insulin content of islet β cells. These results demonstrate a unique strategy for liberating GLP-1 from directly within the target organ and highlight the potential for up-regulating islet GLP-1 production as a means of treating diabetes.

Pax6 and Pdx1 are required for production of glucose-dependent insulinotropic polypeptide in proglucagon-expressing L cells

Glucose-dependent insulinotropic polypeptide (GIP) and glucagon-like peptide-1 (GLP-1) are incretin hormones that play important roles in maintaining glucose homeostasis and are being actively pursued as novel therapeutic agents for diabetes. GIP is produced by dispersed enteroendocrine cells and interestingly at times is coexpressed with GLP-1. We sought to determine the factors that selectively define GIP- vs. GLP-1-expressing cells. We performed comparative immunostaining of Pax6 and Pdx1 in GIP- and GLP-1-secreting cells. We investigated whether Pax6 and Pdx1 activate the human GIP promoter in control IEC-6 cells and GIP-expressing STC-1 cells. EMSA was performed to assess the binding of these transcription factors to the GIP promoter. Pax6 and Pdx1 consistently colocalized in GIP-immunoreactive cells. Cells that coexpress GIP and GLP-1 were Pax6 and Pdx1 positive, whereas cells expressing only GLP-1 were Pax6 positive but did not express Pdx1. GIP promoter activity was enhanced in IEC-6 cells by exogenous Pax6 or Pdx1 and diminished in STC-1 cells by inhibition of endogenous Pax6 or Pdx1 by dominant-negative forms. Promoter truncation analysis revealed a major loss of promoter activity when the sequence between -184 to -145 bp was deleted. EMSA studies indicated that Pax6 and Pdx1 bind to this proximal sequence of the human GIP promoter. Our findings indicate that concomitant expression of Pax6 and Pdx1 is important for GIP expression. Our results also suggest that the presence of Pdx1 defines whether GLP-1-expressing gastrointestinal L cells also coexpress GIP.

Harnessing the gut to treat diabetes

Abstract:  The gut contains one of the largest stem cell populations in the body, yet has been largely overlooked as a source of potentially therapeutic cells. The stem cells reside in the crypts located at the base of the protruding villi, reproduce themselves, and repopulate the gut lining as differentiated cells are sloughed off into the lumen. Some studies have demonstrated that gut stem cells can be isolated and maintained in culture, but the field is currently hampered by the lack of clear markers for these cells. Nevertheless, the relative accessibility of the cells and the similar pathways of differentiation of both intestinal and pancreatic endocrine cells make the gut an attractive potential source of cells to treat diabetes. In particular, it may be possible to recapitulate islet development by the introduction of specific factors to gut stem cells. Alternatively, gut endocrine cells might be coaxed to produce insulin and secrete it into the blood in a meal‐responsive manner. Several investigations support the feasibility of both approaches as novel potential therapies for diabetes. Utilizing a patients own gut cells to re‐establish endogenous meal‐regulated insulin secretion could represent an attractive approach to ultimately cure diabetes.

Treatment of diabetes by transplantation of drug-inducible insulin-producing gut cells

Most patients with type 1 diabetes rely on multiple daily insulin injections to maintain blood glucose control. However, insulin injections carry the risk of inducing hypoglycemia and do not eliminate diabetic complications. We sought to develop and evaluate a regulatable cell-based system for delivery of insulin to treat diabetes. We generated two intestinal cell lines in which human insulin expression is controlled by mifepristone. Insulin mRNA expression was dependent on the mifepristone dose and incubation time and cells displayed insulin and C-peptide immunoreactivity and glucose-induced insulin release following mifepristone treatment. Cell transplantation followed by mifepristone administration reversed streptozotocin (STZ)-induced diabetes in mice, and this effect was dependent on the mifepristone dose delivered. These data support the notion that engineering regulatable insulin expression within a cell already equipped for regulated secretion may be efficacious for the treatment of insulin-dependent diabetes.

488. Development of a Robust Non-Viral Gene Delivery System for Gut Mucosal Cells

The gastrointestinal tract is the largest immune and endocrine organ in the body, and represents an attractive target organ for gene therapy. With a robust gene delivery system for mucosal cells, immune-modulatory proteins can be delivered locally to the gut, thus reducing the potential for systemic side effects. Furthermore, given the accessibility of the gut via the oral route, it is conceivable that a “gene pill” can be formulated to enable oral delivery of a wide range of therapeutic proteins.We report the development of a gene delivery system optimized for gut mucosal cells. The foundation of this gene delivery platform is a novel polymer, synthesized by conjugating arginine (R) and gluconic acid (GA) to low molecular weight poly-glucosamine. We demonstrated by cytotoxicity assay that this novel polymer (DDX) is biocompatible and non-toxic. Polyplex nanoparticles made from DDX containing plasmid DNA have a Z-avg diameter of ~100 nm and ζ of ~ +30 mV. In vitro transfections using DDX as a DNA carrier revealed unexpected synergistic effects, compared to mono-conjugated (R or GA) poly-glucosamine controls. In vivo intramuscular and intrarectal administration in mice also demonstrated superior gene transfection with DDX polyplex relative to R-conjugated and non-conjugated poly-glucosamine polyplexes.To demonstrate efficacy using DDX as a carrier to concentrate expression of the human IL-10 gene to the gut, we assessed the effectiveness of delivery by enema as a treatment for colitis in the dextran sulfate sodium-induced murine colitis model. Treated mice showed improved body weight recovery: (AUC: 19.3 for EG-12-treated vs 7.2 for control-treated, p=0.01). Additionally, the expression levels of pro-inflammatory cytokines (IL-1β, IL-6, IL-17α and TNF-α) in colon tissue of EG-12-treated mice were significantly lower than control-treated animals. Furthermore, localized delivery of IL-10 to the gut of NOD mice using the same gene delivery platform led to significant reduction in diabetes incidence, accompanied by increased regulatory T-cells and decreased activation of insulin-reactive T-cells in the mesenteric lymph nodes. Taken together, our results indicate that our novel gene transfer system can effectively deliver and concentrate immune-modulatory proteins to gut mucosal cells.We aim to formulate the polyplex for oral administration to achieve transient, but renewable, protein production in the gut. This platform may enable delivery of proteins into the circulation via the oral route. To this end, we have developed processes to convert the liquid formulation into dry powder by both spray drying or lyophilization, followed by compression into tablets or filled into capsules. Dissolution testing of these tablets and capsules in water revealed that the nanoparticles remain intact and biologically active. Preliminary studies in rats demonstrated detectable circulating IL-10 after oral administration of non-enteric coated capsules. With enteric coating, a platform capable of oral dosage of protein drugs may be made possible.