Establishing a Large-Animal Model for In Vivo Reprogramming of Bile Duct Cells into Insulin-Secreting Cells to Treat Diabetes.

Type 1 diabetes manifests as autoimmune destruction of beta cells requiring metabolic management with an exogenous replacement of insulin, either by repeated injection of recombinant insulin or by transplantation of allogeneic islets from cadaveric donors. Both of these approaches have severe limitations. Repeated insulin injection requires intensive blood glucose monitoring, is expensive, and is associated with decreased quality-of-life measures. Islet transplantation, while highly effective, is severely limited by shortage of donor organs. Clinical translation of beta cells derived from pluripotent stem cells is also not yet a reality, and alternative approaches to solving the replacement of lost beta cell function are required. In vivo direct reprogramming offers an attractive approach to generating new endogenous insulin-secreting cells by permanently altering the phenotype of somatic cells after transient expression of transcription factors. Previously, we have successfully restored control of blood glucose in diabetic mice by reprogramming liver cells into glucose-sensitive insulin-secreting cells after the transient, simultaneous delivery of three transcription factors (Pdx1, Ngn3, and MafA) to the liver of diabetic mice, using an adenoviral vector (Ad-PNM). Establishing a clinically relevant, large-animal model is a critical next step in translating this approach beyond the proof-of-principle stage in rodents and allowing investigation of vector design, dose and delivery, host response to vector infusion, and establishment of suitable criteria for measuring safety and efficacy. In this feasibility study we infused Ad-PNM into the liver of three diabetic cynomolgus macaques via portal vein catheter. Vector presence and cargo gene and protein expression were detected in liver tissue after infusion with no adverse effects. Refinement of immune suppression significantly extended the period of exogenous PNM expression. This pilot study establishes the suitability of this large-animal model to examine the translation of this approach for treating diabetes.

Reprogramming of various cell types to a beta-like state by Pdx1, Ngn3 and MafA.

The three transcription factors, PDX1, NGN3 and MAFA, are very important in pancreatic development. Overexpression of these three factors can reprogram both pancreatic exocrine cells and SOX9-positive cells of the liver into cells resembling pancreatic beta cells. In this study we investigate whether other cell types can be reprogrammed. Eight cell types are compared and the results are consistent with the idea that reprogramming occurs to a greater degree for developmentally related cells (pancreas, liver) than for other types, such as fibroblasts. Using a line of mouse hepatocyte-derived cells we screened 13 compounds for the ability to increase the yield of reprogrammed cells. Three are active and when used in combination they can increase the yield of insulin-immunopositive cells by a factor of six. These results should contribute to the eventual ability to develop a new cure for diabetes based on the ability to reprogram other cells in the body to a beta cell phenotype.

Stage specific reprogramming of mouse embryo liver cells to a beta cell-like phenotype.

We show that cultures of mouse embryo liver generate insulin-positive cells when transduced with an adenoviral vector encoding the three genes: Pdx1, Ngn3 and MafA (Ad-PNM). Only a proportion of transduced cells become insulin-positive and the highest yield occurs in the period E14-16, declining at later stages. Insulin-positive cells do not divide further although they can persist for several weeks. RT-PCR analysis of their gene expression shows the upregulation of a whole battery of genes characteristic of beta cells including upregulation of the endogenous counterparts of the input genes. Other features, including a relatively low insulin content, the expression of genes for other pancreatic hormones, and the fact that insulin secretion is not glucose-sensitive, indicate that the insulin-positive cells remain immature. The origin of the insulin-positive cells is established both by co-immunostaining for α-fetoprotein and albumin, and by lineage tracing for Sox9, which is expressed in the ductal plate cells giving rise to biliary epithelium. This shows that the majority of insulin-positive cells arise from hepatoblasts with a minority from the ductal plate cells.

In vivo reprogramming of Sox9+ cells in the liver to insulin-secreting ducts.

In embryonic development, the pancreas and liver share developmental history up to the stage of bud formation. Therefore, we postulated that direct reprogramming of liver to pancreatic cells can occur when suitable transcription factors are overexpressed. Using a polycistronic vector we misexpress Pdx1, Ngn3, and MafA in the livers of NOD-SCID mice rendered diabetic by treatment with streptozotocin (STZ). The diabetes is relieved long term. Many ectopic duct-like structures appear that express a variety of ß-cell markers, including dense core granules visible by electron microscopy (EM). Use of a vector also expressing GFP shows that the ducts persist long after the viral gene expression has ceased, indicating that this is a true irreversible cell reprogramming event. We have recovered the insulin(+) cells by cell sorting and shown that they display glucose-sensitive insulin secretion. The early formed insulin(+) cells can be seen to coexpress SOX9 and are also labeled in mice lineage labeled for Sox9 expression. SOX9(+) cells are normally found associated with small bile ducts in the periportal region, indicating that the duct-like structures arise from this source. This work confirms that developmentally related cells can be reprogrammed by suitable transcription factors and also suggests a unique therapy for diabetes.

Reprogramming of pancreatic exocrine cells towards a beta (ß) cell character using Pdx1, Ngn3 and MafA.

Pdx1 (pancreatic and duodenal homeobox 1), Ngn3 (neurogenin 3) and MafA (v-maf musculoaponeurotic fibrosarcoma oncogene family, protein A) have been reported to bring about the transdifferentiation of pancreatic exocrine cells to beta (ß) cells in vivo. We have investigated the mechanism of this process using a standard in vitro model of pancreatic exocrine cells, the rat AR42j-B13 cell line. We constructed a new adenoviral vector encoding all three genes, called Ad-PNM (adenoviral Pdx1, Ngn3, MafA construct). When introduced into AR42j-B13 cells, Ad-PNM caused a rapid change to a flattened morphology and a cessation of cell division. The expression of exocrine markers is suppressed. Both insulin genes are up-regulated as well as a number of transcription factors normally characteristic of beta cells. At the chromatin level, histone tail modifications of the Pdx1, Ins1 (insulin 1) and Ins2 (insulin 2) gene promoters are shifted in a direction associated with gene activity, and the level of DNA CpG methylation is reduced at the Ins1 promoter. The transformed cells secrete insulin and are capable of relieving diabetes in streptozotocin-treated NOD-SCID (non-obese diabetic severe combined immunodeficiency) mice. However the transformation is not complete. The cells lack expression of several genes important for beta cell function and they do not show glucose-sensitive insulin secretion. We conclude that, for this exocrine cell model, although the transformation is dramatic, the reprogramming is not complete and lacks critical aspects of the beta cell phenotype.

Adiponectin translation is increased by the PPARgamma agonists pioglitazone and omega-3 fatty acids.

Adiponectin, made exclusively by adipocytes, is a 30-kDa secretory protein assembled posttranslationally into low-molecular weight, middle-molecular weight, and high-molecular weight homo-oligomers. PPARgamma ligand thiozolidinediones, which are widely used in the treatment of type II diabetes, increase adiponectin levels. PPARgamma also has several putative ligands that include fatty acid derivatives. Overnight treatment of rat adipocytes with pioglitazone, docosahexaenoic acid (DHA), or eicosapentaenoic acid (EPA) triggered a twofold increase in the synthesis and secretion of HMW adiponectin, and this increase was blocked by the addition of PPARgamma inhibitor GW-9662. Inhibition of glycosylation using 2,2'-dipyridyl decreased the synthesis of high-molecular weight adiponectin by pioglitazone, EPA, and DHA, but there was increased secretion of trimeric adiponectin resulting from increased translation. Although pioglitazone, DHA, and EPA increased adiponectin synthesis by more than 60%, there was no increase in total protein synthesis and no corresponding change in adiponectin mRNA expression, indicating the upregulation of translation. We examined the possibility of transacting factors in the cytoplasmic extracts from adipocytes treated with pioglitazone or DHA. In vitro translation of adiponectin mRNA was inhibited by S-100 fraction of control adipocytes and increased by S-100 extracts from adipocytes treated with pioglitazone or DHA. Consistent with this observation, both pioglitazone and DHA treatments increased the association of adiponectin mRNA with the heavier polysome fractions. Together, these data suggest that pioglitazone and the fish oils DHA or EPA are PPARgamma agonists in adipocytes with regard to adiponectin expression, and the predominant mode of adiponectin stimulation is via an increase in translation.

Calcium is involved in formation of high molecular weight adiponectin.

BACKGROUND: Adiponectin, an adipocyte-specific secretory protein, is known to circulate as different isoforms in the blood stream. METHODS: Using sucrose gradients and Western blotting on nondenaturing gels, adiponectin isoforms were examined in human serum, plasma, adipose tissue, and cells. The medium from human adipose tissue and human and mouse adipocytes were also examined for changes in isoform formation upon treatment with EGTA. RESULTS: Comparison of adiponectin complexes revealed distinct differences in distribution of high molecular weight (HMW) forms between human serum and plasma, with an apparent difference in molecular weight. Variation in molecular weight suggested a probable dissociation of the HMW isoforms in the presence of EDTA in the plasma. Examination of human serum samples treated with EDTA or EGTA showed a partial dissociation of the HMW isoform, while the addition of excess calcium, but not magnesium, to human plasma resulted in partial restoration of HMW adiponectin. When human adipose tissue-secreted adiponectin was treated with EGTA, there was a decrease in the HMW isoform by 61% (+/- 1.89%) and a corresponding increase in low molecular weight (LMW) and middle molecular weight (MMW) isoforms, compared to untreated samples. Analysis of mouse and human adipocytes also showed a reduction in HMW isoforms with a corresponding increase in MMW and LMW isoforms upon treatment with EGTA. The Simpson-Golabi-Behmel syndrome (SGBS) human adipocyte cell line, which primarily synthesizes LMW isoforms, produced increasing amounts of HMW adiponectin upon treatment with calcium in a dose-dependent manner. CONCLUSION: These data indicate that calcium promotes the formation of HMW adiponectin, and calcium sequestration decreases HMW adiponectin. Because of the importance of HMW adiponectin in insulin sensitivity, these data demonstrate the importance of assay conditions and sample preparation in the measurement of adiponectin isoforms.

Pioglitazone increases secretion of high-molecular-weight adiponectin from adipocytes.

Adiponectin is an adipocyte-derived serum protein that plays important roles in energy homeostasis, obesity, and insulin sensitivity. Using sucrose gradients and Western blotting of nondenaturing gels, we examined the adiponectin isoforms secreted from human adipose tissue, human and mouse adipocytes, and cell lines in response to pioglitazone added in vitro. The predominant form secreted from adipose tissue in vitro was the high-molecular-weight (HMW) isoform, with small amounts of low-molecular-weight (LMW) forms present. The addition of pioglitazone (1-3 micromM) in vitro increased the secretion of the HMW isoform, with no significant effect on the other isoforms. Human adipose tissue was also examined for changes in adiponectin mRNA levels upon pioglitazone treatment. No difference was detected, suggesting that the effect of pioglitazone is not at the transcriptional level but, rather, at a posttranscriptional phase of the secretory pathway. Additional experiments were conducted to determine whether adiponectin expression was mechanistically similar in other adipose cells. Examination of primary human adipocytes revealed an increase in intracellular HMW isoform with a decline in LMW forms following pioglitazone treatment, with a corresponding increase in the secreted HMW form. Similar results were observed with primary mouse adipocytes, 3T3-F422A cells, and SGBS human adipocyte cells, although differences in the distribution of HMW and LMW isoforms were apparent between cell types. Although there are differences in isoforms between species, in all cases pioglitazone served to increase the secretion of the HMW form of adiponectin.