Rhonda D. Wideman

Production of Functional Glucagon-Secreting α-Cells From Human Embryonic Stem Cells

OBJECTIVE Differentiation of human embryonic stem (hES) cells to fully developed cell types holds great therapeutic promise. Despite significant progress, the conversion of hES cells to stable, fully differentiated endocrine cells that exhibit physiologically regulated hormone secretion has not yet been achieved. Here we describe an efficient differentiation protocol for the in vitro conversion of hES cells to functional glucagon-producing α- cells. RESEARCH DESIGN AND METHODS Using a combination of small molecule screening and empirical testing, we developed a six-stage differentiation protocol for creating functional α-cells. An extensive in vitro and in vivo characterization of the differentiated cells was performed. RESULTS A high rate of synaptophysin expression (>75%) and robust expression of glucagon and the α-cell transcription factor ARX was achieved. After a transient polyhormonal state in which cells coexpress glucagon and insulin, maturation in vitro or in vivo resulted in depletion of insulin and other β-cell markers with concomitant enrichment of α-cell markers. After transplantation, these cells secreted fully processed, biologically active glucagon in response to physiologic stimuli including prolonged fasting and amino acid challenge. Moreover, glucagon release from transplanted cells was sufficient to reduce demand for pancreatic glucagon, resulting in a significant decrease in pancreatic α-cell mass. CONCLUSIONS These results indicate that fully differentiated pancreatic endocrine cells can be created via stepwise differentiation of hES cells. These cells may serve as a useful screening tool for the identification of compounds that modulate glucagon secretion as well as those that promote the transdifferentiation of α-cells to β-cells.

Glucose-Dependent Insulinotropic Polypeptide Is Expressed in Pancreatic Islet α-Cells and Promotes Insulin Secretion

BACKGROUND & AIMS Glucose-dependent insulinotropic polypeptide (GIP) and the proglucagon product glucagon-like peptide-1 (GLP-1) are gastrointestinal hormones that are released in response to nutrient intake and promote insulin secretion. Interestingly, a subset of enteroendocrine cells express both GIP and GLP-1. We sought to determine whether GIP also might be co-expressed with proglucagon in pancreatic alpha-cells. METHODS We assessed GIP expression via reverse-transcription polymerase chain reaction, in situ hybridization, and immunohistochemistry. We developed a novel bioassay to measure GIP release from isolated islets, compared the biological activities of full-length and truncated GIP, and assessed the impact of immunoneutralization of islet GIP on glucose-stimulated insulin secretion in isolated islets. RESULTS GIP messenger RNA was present in mouse islets; GIP protein localized to islet alpha-cells of mouse, human, and snake pancreas, based on immunohistochemical analyses. However, using a C-terminal GIP antibody, immunoreactivity was detected in islets from prohormone convertase (PC) 2 knockout but not wild-type mice. Bioactive GIP was secreted from mouse and human islets after arginine stimulation. In the perfused mouse pancreas, GIP(1-42) and amidated GIP(1-30) had equipotent insulinotropic actions. Finally, immunoneutralization of GIP secreted by isolated islets decreased glucose-stimulated insulin secretion. CONCLUSIONS GIP is expressed in and secreted from pancreatic islets; in alpha-cells, PC2 processes proGIP to yield a truncated but bioactive form of GIP that differs from the PC1/3-derived form from K-cells. Islet-derived GIP promotes islet glucose competence and also could support islet development and/or survival.

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.

Leptin Therapy Reverses Hyperglycemia in Mice With Streptozotocin-Induced Diabetes, Independent of Hepatic Leptin Signaling

OBJECTIVE Leptin therapy has been found to reverse hyperglycemia and prevent mortality in several rodent models of type 1 diabetes. Yet the mechanism of leptin-mediated reversal of hyperglycemia has not been fully defined. The liver is a key organ regulating glucose metabolism and is also a target of leptin action. Thus we hypothesized that exogenous leptin administered to mice with streptozotocin (STZ)-induced diabetes reverses hyperglycemia through direct action on hepatocytes. RESEARCH DESIGN AND METHODS After the induction of diabetes in mice with a high dose of STZ, recombinant mouse leptin was delivered at a supraphysiological dose for 14 days by an osmotic pump implant. We characterized the effect of leptin administration in C57Bl/6J mice with STZ-induced diabetes and then examined whether leptin therapy could reverse STZ-induced hyperglycemia in mice in which hepatic leptin signaling was specifically disrupted. RESULTS Hyperleptinemia reversed hyperglycemia and hyperketonemia in diabetic C57Bl/6J mice and dramatically improved glucose tolerance. These effects were associated with reduced plasma glucagon and growth hormone levels and dramatically enhanced insulin sensitivity, without changes in glucose uptake by skeletal muscle. Leptin therapy also ameliorated STZ-induced hyperglycemia and hyperketonemia in mice with disrupted hepatic leptin signaling to a similar extent as observed in wild-type littermates with STZ-induced diabetes. CONCLUSIONS These observations reveal that hyperleptinemia reverses the symptoms of STZ-induced diabetes in mice and that this action does not require direct leptin signaling in the liver.

A Switch From Prohormone Convertase (PC)-2 to PC1/3 Expression in Transplanted α-Cells Is Accompanied by Differential Processing of Proglucagon and Improved Glucose Homeostasis in Mice

OBJECTIVE—Glucagon, which raises blood glucose levels by stimulating hepatic glucose production, is produced in α-cells via cleavage of proglucagon by prohormone convertase (PC)-2. In the enteroendocrine L-cell, proglucagon is differentially processed by the alternate enzyme PC1/3 to yield glucagon-like peptide (GLP)-1, GLP-2, and oxyntomodulin, which have blood glucose–lowering effects. We hypothesized that alteration of PC expression in α-cells might convert the α-cell from a hyperglycemia-promoting cell to one that would improve glucose homeostasis. RESEARCH DESIGN AND METHODS—We compared the effect of transplanting encapsulated PC2-expressing αTC-1 cells with PC1/3-expressing αTCΔPC2 cells in normal mice and low-dose streptozotocin (STZ)-treated mice. RESULTS—Transplantation of PC2-expressing α-cells increased plasma glucagon levels and caused mild fasting hyperglycemia, impaired glucose tolerance, and α-cell hypoplasia. In contrast, PC1/3-expressing α-cells increased plasma GLP-1/GLP-2 levels, improved glucose tolerance, and promoted β-cell proliferation. In GLP-1R−/− mice, the ability of PC1/3-expressing α-cells to improve glucose tolerance was attenuated. Transplantation of PC1/3-expressing α-cells prevented STZ-induced hyperglycemia by preserving β-cell area and islet morphology, possibly via stimulating β-cell replication. However, PC2-expressing α-cells neither prevented STZ-induced hyperglycemia nor increased β-cell proliferation. Transplantation of αTCΔPC2, but not αTC-1 cells, also increased intestinal epithelial proliferation. CONCLUSIONS—Expression of PC1/3 rather than PC2 in α-cells induces GLP-1 and GLP-2 production and converts the α-cell from a hyperglycemia-promoting cell to one that lowers blood glucose levels and promotes islet survival. This suggests that alteration of proglucagon processing in the α-cell may be therapeutically useful in the context of diabetes.

A Switch from PC2 to PC1/3 Expression in Transplanted α-cells is Accompanied by Differential Processing of Proglucagon and Improved Glucose Homeostasis in Mice

OBJECTIVE—Glucagon, which raises blood glucose levels by stimulating hepatic glucose production, is produced in α-cells via cleavage of proglucagon by prohormone convertase (PC)-2. In the enteroendocrine L-cell, proglucagon is differentially processed by the alternate enzyme PC1/3 to yield glucagon-like peptide (GLP)-1, GLP-2, and oxyntomodulin, which have blood glucose–lowering effects. We hypothesized that alteration of PC expression in α-cells might convert the α-cell from a hyperglycemia-promoting cell to one that would improve glucose homeostasis. RESEARCH DESIGN AND METHODS—We compared the effect of transplanting encapsulated PC2-expressing αTC-1 cells with PC1/3-expressing αTCΔPC2 cells in normal mice and low-dose streptozotocin (STZ)-treated mice. RESULTS—Transplantation of PC2-expressing α-cells increased plasma glucagon levels and caused mild fasting hyperglycemia, impaired glucose tolerance, and α-cell hypoplasia. In contrast, PC1/3-expressing α-cells increased plasma GLP-1/GLP-2 levels, improved glucose tolerance, and promoted β-cell proliferation. In GLP-1R−/− mice, the ability of PC1/3-expressing α-cells to improve glucose tolerance was attenuated. Transplantation of PC1/3-expressing α-cells prevented STZ-induced hyperglycemia by preserving β-cell area and islet morphology, possibly via stimulating β-cell replication. However, PC2-expressing α-cells neither prevented STZ-induced hyperglycemia nor increased β-cell proliferation. Transplantation of αTCΔPC2, but not αTC-1 cells, also increased intestinal epithelial proliferation. CONCLUSIONS—Expression of PC1/3 rather than PC2 in α-cells induces GLP-1 and GLP-2 production and converts the α-cell from a hyperglycemia-promoting cell to one that lowers blood glucose levels and promotes islet survival. This suggests that alteration of proglucagon processing in the α-cell may be therapeutically useful in the context of diabetes.

Clinical Application of Glucagon-Like Peptide 1 Receptor Agonists for the Treatment of Type 2 Diabetes Mellitus

Glucagon-like peptide 1 (GLP-1) is secreted from enteroendocrine L-cells in response to oral nutrient intake and elicits glucose-stimulated insulin secretion while suppressing glucagon secretion. It also slows gastric emptying, which contributes to decreased postprandial glycemic excursions. In the 1990s, chronic subcutaneous infusion of GLP-1 was found to lower blood glucose levels in patients with type 2 diabetes. However, GLP-1s very short half-life, arising from cleavage by the enzyme dipeptidyl peptidase 4 (DPP-4) and glomerular filtration by the kidneys, presented challenges for clinical use. Hence, DPP-4 inhibitors were developed, as well as several GLP-1 analogs engineered to circumvent DPP-4-mediated breakdown and/or rapid renal elimination. Three categories of GLP-1 analogs, are being developed and/or are in clinical use: short-acting, long-acting, and prolonged-acting GLP-1 analogs. Each class has different plasma half-lives, molecular size, and homology to native GLP-1, and consequently different characteristic effects on glucose metabolism. In this article, we review current clinical data derived from each class of GLP-1 analogs, and consider the clinical effects reported for each category in recent head to head comparison studies. Given the relatively brief clinical history of these compounds, we also highlight several important efficacy and safety issues which will require further investigation.

Mining incretin hormone pathways for novel therapies

The incretin hormones, glucose-dependent insulinotropic polypeptide (GIP) and glucagon-like peptide-1 (GLP-1), are produced predominantly by enteroendocrine cells and have multiple blood glucose-lowering effects. Recent years have seen a surge of interest in understanding the basic physiology and pathophysiology of incretins and in applying this knowledge to the treatment of diabetes and obesity. Considerable gains have been made in elucidating the mechanisms controlling incretin secretion, and there is growing evidence to suggest that incretins might be involved in the rapid reversal of diabetes observed in gastric bypass patients. Here, we review these recent advances and outline the multiple strategies being pursued to exploit the potential therapeutic benefits of GIP and GLP-1.

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.

Cellular reprogramming of human amniotic fluid cells to express insulin.

Islet transplantation represents a potential cure for type 1 diabetes; however, a lack of sufficient donor material limits its clinical use. To address the shortfall of islet availability, surrogate insulin-producing cells are sought. Studies suggest that human amniotic fluid (hAF) contains multipotent progenitor cells capable of differentiating to all three germ layers. Here, we used high-content, live-cell imaging to assess the ability to reprogram hAF cells towards a beta cell phenotype. A fluorescent reporter system was developed where DsRed express (DSRE) expression is driven by the human insulin promoter. Using integrative lentiviral technology, we created stable reporter hAF cells that could be routinely monitored for insulin promoter activation. These cells were subjected to combinatorial high-content screening using adenoviral-mediated expression of up to six transcription factors important for beta cell development. Cells were monitored for DSRE expression which revealed an optimal combination of the transcription factors required to induce insulin gene expression in hAF cells. These optimally induced cells were examined for expression of additional beta cell transcription factors and proteins involved in glucose sensing and insulin processing. RT-qPCR revealed very low level expression of insulin that was ultimately insufficient to reverse streptozotocin-induced diabetes following sub-capsular kidney transplantation. High-content, live-cell imaging using fluorescent reporter cells provides a convenient method for repeated assessment of cellular reprogramming. hAF cells could be reprogrammed to express key beta cell proteins, however insulin gene expression was insufficient to reverse hyperglycemia in diabetic animals.