Specific Reprogramming of Alpha Cells to Insulin-Producing Cells by Short Glucagon Promoter-Driven Pdx1 and MafA

Endogenous reprogramming of pancreas-derived non-beta cells into insulin-producing cells is a promising approach to treat type 1 diabetes (T1D). One strategy that has yet to be explored is the specific delivery of insulin-producing essential genes, Pdx1 and MafA, to pancreatic alpha cells to reprogram the cells into insulin-producing cells in an adult pancreas. In this study, we utilized an alpha cell-specific glucagon (GCG) promoter to drive Pdx1 and MafA transcription factors to reprogram alpha cells to insulin-producing cells in chemically induced and autoimmune diabetic mice. Our results showed that a combination of a short glucagon-specific promoter with AAV serotype 8 can be used to successfully deliver Pdx1 and MafA into alpha cells in the mouse pancreas. Pdx1 and MafA expression specifically in alpha cells was also able to correct hyperglycemia in both induced and autoimmune diabetic mice. With this technology, targeted gene specificity and reprogramming were accomplished with an alpha-specific promotor combined with an AAV-specific serotype and provide an initial basis to develop a novel therapy for the treatment of T1D.

Specific Reprogramming of Alpha Cells to Insulin-Producing Cells by Short Glucagon Promoter-Driven Pdx1 and MafA

Endogenous reprogramming of pancreas-derived non-beta cells into insulin-producing cells is a promising approach to treat type 1 diabetes (T1D). One strategy that has yet to be explored is the specific delivery of insulin-producing essential genes, Pdx1 and MafA, to pancreatic alpha cells to reprogram the cells into insulin-producing cells in an adult pancreas. In this study, we utilized an alpha cell-specific glucagon (GCG) promoter to drive Pdx1 and MafA transcription factors to reprogram alpha cells to insulin-producing cells in chemically induced and autoimmune diabetic mice. Our results showed that a combination of a short glucagon-specific promoter with AAV serotype 8 can be used to successfully deliver Pdx1 and MafA into alpha cells in the mouse pancreas. Pdx1 and MafA expression specifically in alpha cells was also able to correct hyperglycemia in both induced and autoimmune diabetic mice. With this technology, targeted gene specificity and reprogramming were accomplished with an alpha-specific promotor combined with an AAV-specific serotype and provide an initial basis to develop a novel therapy for the treatment of T1D.

Arginine-vasopressin mediates counter-regulatory glucagon release and is diminished in type 1 diabetes

Insulin-induced hypoglycemia is a major barrier to the treatment of type-1 diabetes (T1D). Accordingly, it is important that we understand the mechanisms regulating the circulating levels of glucagon - the body's principal blood glucose-elevating hormone which is secreted from alpha-cells of the pancreatic islets. Varying glucose over the range of concentrations that occur physiologically between the fed and fuel-deprived states (from 8 to 4 mM) has no significant effect on glucagon secretion in the perfused mouse pancreas or in isolated mouse islets (in vitro) and yet associates with dramatic changes in plasma glucagon in vivo. The identity of the systemic factor(s) that stimulates glucagon secretion remains unknown. Here, we show that arginine-vasopressin (AVP), secreted from the posterior pituitary, stimulates glucagon secretion. Glucagon-secreting alpha-cells express high levels of the vasopressin 1b receptor gene (Avpr1b). Activation of AVP neurons in vivo increased circulating copeptin (the C-terminal segment of the AVP precursor peptide, a stable surrogate marker of AVP) and increased blood glucose; effects blocked by pharmacological antagonism of either the glucagon receptor or vasopressin 1b receptor. AVP also mediates the stimulatory effects of hypoglycemia produced by exogenous insulin and 2-deoxy-D-glucose on glucagon secretion. We show that the A1/C1 neurons of the medulla oblongata drive AVP neuron activation in response to insulin-induced hypoglycemia. Exogenous injection of AVP in vivo increased cytoplasmic Ca2+ in alpha-cells (implanted into the anterior chamber of the eye) and glucagon release. Hypoglycemia also increases circulating levels of AVP in humans and this hormone stimulates glucagon secretion from isolated human islets. In patients with T1D, hypoglycemia failed to increase both plasma copeptin and glucagon levels. These findings suggest that AVP is a physiological systemic regulator of glucagon secretion and that this mechanism becomes impaired in T1D.

Immunolocalization of the EDWM-Protein Indicates a Matrix Role in Cornification of Lizard Epidermis

During epidermal differentiation in the scales of lizards and snakes, from the basal layer beta- and later alpha-keratinocytes are generated to form beta-and alpha-corneous layers. In the lizard Anolis carolinensis, minor proteins derived from the EDC (Epidermal Differentiation Complex) are added to the main constituent proteins, IFKs (Intermediate Filament Keratins) and CBPs (Corneous Beta Proteins, formerly indicated as beta keratins). One of these proteins that previous studies showed to be exclusively expressed in the skin, EDWM (EDC protein containing high GSRC amino acids) is rich in cysteine and arginine, amino acids that form numerous –S–S– and electro-static chemical bonds in the corneous material. Light and electron microscopy immunolbeling for EDWM show a diffuse localization in differentiating beta-cells and in some alpha-cells, in particular those of the clear-layer, involved in epidermal shedding. The study suggests that EDWM may function as a matrix protein that binds to IFKs and CBPs, contributing to the formation of the specific corneous material present in beta- and alpha-corneous layers. In particular, its higher immunolocalization in the maturing clear layer indicates that this protein is important for its differentiation and epidermal shedding in A. carolinensis and likely also in other lepidosaurian reptiles.

Specific Reprogramming of Alpha Cells to Insulin-Producing Cells by Short Glucagon Promoter-Driven Pdx1 and MafA

Endogenous reprogramming of pancreas-derived non-beta cells into insulin-producing cells is a promising approach to treat type 1 diabetes (T1D). One strategy that has yet to be explored is the specific delivery of insulin-producing essential genes, Pdx1 and MafA, to pancreatic alpha cells to reprogram the cells into insulin-producing cells in an adult pancreas. In this study, we utilized an alpha cell-specific glucagon (GCG) promoter to drive Pdx1 and MafA transcription factors to reprogram alpha cells to insulin-producing cells in chemically induced and autoimmune diabetic mice. Our results showed that a combination of a short glucagon-specific promoter with AAV serotype 8 can be used to successfully deliver Pdx1 and MafA into alpha cells in the mouse pancreas. Pdx1 and MafA expression specifically in alpha cells was also able to correct hyperglycemia in both induced and autoimmune diabetic mice. With this technology, targeted gene specificity and reprogramming were accomplished with an alpha-specific promotor combined with an AAV-specific serotype and provide an initial basis to develop a novel therapy for the treatment of T1D.

The Role of Pancreatic Alpha Cells and Endothelial Cells in the Reduction of Oxidative Stress in Pseudoislets

Pancreatic beta cells have inadequate levels of antioxidant enzymes, and the damage induced by oxidative stress poses a challenge for their use in a therapy for patients with type 1 diabetes. It is known that the interaction of the pancreatic endocrine cells with support cells can improve their survival and lead to less vulnerability to oxidative stress. Here we investigated alpha (alpha TC-1), beta (INS1E) and endothelial (HUVEC) cells assembled into aggregates known as pseudoislets as a model of the pancreatic islets of Langerhans. We hypothesised that the coculture of alpha, beta and endothelial cells would be protective against oxidative stress. First, we showed that adding endothelial cells decreased the percentage of oxidative stress-positive cells. We then asked if the number of endothelial cells or the size (number of cells) of the pseudoislet could increase the protection against oxidative stress. However, no additional benefit was observed by those changes. On the other hand, we identified a potential supportive effect of the alpha cells in reducing oxidative stress in beta and endothelial cells. We were able to link this to the incretin glucagon-like peptide-1 (GLP-1) by showing that the absence of alpha cells in the pseudoislet caused increased oxidative stress, but the addition of GLP-1 could restore this. Together, these results provide important insights into the roles of alpha and endothelial cells in protecting against oxidative stress.

FAILURE OF PANCREATIC ALPHA CELLS TO RESPOND TO HYPOGLYCEMIA IS LINKED TO IMPAIRED GLUTAMATE RECEPTOR SIGNALING IN DIABETES

Glucagon secretion from the pancreatic alpha cells is crucial to prevent hypoglycemia. People with type 1 diabetes, however, lose this glucoregulatory mechanism and are susceptible to dangerous insulin treatment-induced hypoglycemia. We established that activating glutamate receptors of the AMPA/kainate type in alpha cells is needed for decreases in glucose levels to elicit full glucagon responses from mouse and human islets. We performed functional studies using living pancreas slices from donors with type 1 diabetes and found that alpha cells had normal glucagon content and responded typically to KCl depolarization, but failed to respond to decreases in glucose concentration and had severely impaired AMPA/kainate receptor signaling. Reactivating residual AMPA/kainate receptor function with the positive allosteric modulators cyclothiazide and aniracetam partially rescued glucagon secretion in response to hypoglycemia. Positive allosteric modulators of AMPA/kainate receptors already approved to treat other conditions could thus be repurposed to prevent hypoglycemia and improve management of diabetes.

High co-expression of the ghrelin and LEAP2 receptor GHSR with pancreatic polypeptide in mouse and human islets

Islets represent an important site of direct action of the hormone ghrelin, with expression of the ghrelin receptor (growth hormone secretagogue receptor; GHSR) having been localized variably to alpha-cells, beta-cells, and/or somatostatin (SST)-secreting delta-cells. To our knowledge, GHSR expression by pancreatic polypeptide (PP)-expressing gamma-cells has not been specifically investigated. Here, histochemical analyses of Ghsr-IRES-Cre X Cre-dependent ROSA26-YFP reporter mice showed 85% of GHSR-expressing islet cells co-express PP, 50% co-express SST, and 47% co-express PP + SST. Analysis of single-cell transcriptomic data from mouse pancreas revealed 95% of Ghsr-expressing cells co-express Ppy, 100% co-express Sst, and 95% co-express Ppy + Sst. This expression was restricted to gamma-cell and delta-cell clusters. Analysis of several single-cell human pancreatic transcriptome datasets revealed 59% of GHSR-expressing cells co-express PPY, 95% co-express SST, and 57% co-express PPY + SST. This expression was prominent in delta-cell and beta-cell clusters, also occurring in other clusters including gamma-cells and alpha-cells. GHSR expression levels were upregulated by type 2 diabetes mellitus in beta-cells. In mice, plasma PP positively correlated with fat mass and with plasma levels of the endogenous GHSR antagonist/inverse agonist LEAP2. Plasma PP also elevated upon LEAP2 and synthetic GHSR antagonist administration. These data suggest that in addition to delta-cells, beta-cells, and alpha-cells, PP-expressing pancreatic cells likely represent important direct targets for LEAP2 and/or ghrelin in both mice and humans.