Pedro Luis Herrera

Conversion of adult pancreatic α-cells to β-cells after extreme β-cell loss

Pancreatic insulin-producing β-cells have a long lifespan, such that in healthy conditions they replicate little during a lifetime. Nevertheless, they show increased self-duplication after increased metabolic demand or after injury (that is, β-cell loss). It is not known whether adult mammals can differentiate (regenerate) new β-cells after extreme, total β-cell loss, as in diabetes. This would indicate differentiation from precursors or another heterologous (non-β-cell) source. Here we show β-cell regeneration in a transgenic model of diphtheria-toxin-induced acute selective near-total β-cell ablation. If given insulin, the mice survived and showed β-cell mass augmentation with time. Lineage-tracing to label the glucagon-producing α-cells before β-cell ablation tracked large fractions of regenerated β-cells as deriving from α-cells, revealing a previously disregarded degree of pancreatic cell plasticity. Such inter-endocrine spontaneous adult cell conversion could be harnessed towards methods of producing β-cells for diabetes therapies, either in differentiation settings in vitro or in induced regeneration.

Conversion of adult pancreatic alpha-cells to beta-cells after extreme beta-cell loss.

Pancreatic insulin-producing β-cells have a long lifespan, such that in healthy conditions they replicate little during a lifetime. Nevertheless, they show increased self-duplication after increased metabolic demand or after injury (that is, β-cell loss). It is not known whether adult mammals can differentiate (regenerate) new β-cells after extreme, total β-cell loss, as in diabetes. This would indicate differentiation from precursors or another heterologous (non-β-cell) source. Here we show β-cell regeneration in a transgenic model of diphtheria-toxin-induced acute selective near-total β-cell ablation. If given insulin, the mice survived and showed β-cell mass augmentation with time. Lineage-tracing to label the glucagon-producing α-cells before β-cell ablation tracked large fractions of regenerated β-cells as deriving from α-cells, revealing a previously disregarded degree of pancreatic cell plasticity. Such inter-endocrine spontaneous adult cell conversion could be harnessed towards methods of producing β-cells for diabetes therapies, either in differentiation settings in vitro or in induced regeneration.

Embryonic endocrine pancreas and mature β cells acquire α and PP cell phenotypes upon Arx misexpression

Aristaless-related homeobox (Arx) was recently demonstrated to be involved in pancreatic alpha cell fate specification while simultaneously repressing the beta and delta cell lineages. To establish whether Arx is not only necessary, but also sufficient to instruct the alpha cell fate in endocrine progenitors, we used a gain-of-function approach to generate mice conditionally misexpressing this factor. Mice with forced Arx expression in the embryonic pancreas or in developing islet cells developed a dramatic hyperglycemia and eventually died. Further analysis demonstrated a drastic loss of beta and delta cells. Concurrently, a remarkable increase in the number of cells displaying alpha cell or, strikingly, pancreatic polypeptide (PP) cell features was observed. Notably, the ectopic expression of Arx induced in embryonic or adult beta cells led to a loss of the beta cell phenotype and a concomitant increase in a number of cells with alpha or PP cell characteristics. Combining quantitative real-time PCR and lineage-tracing experiments, we demonstrate that, in adult mice, the misexpression of Arx, rather than its overexpression, promotes a conversion of beta cells into glucagon- or PP-producing cells in vivo. These results provide important insights into the complex mechanisms underlying proper pancreatic endocrine cell allocation and cell identity acquisition.

Insulin Signaling in α-cells Modulates Glucagon Secretion in vivo

Glucagon plays an important role in glucose homeostasis by regulating hepatic glucose output in both normo- and hypoglycemic conditions. In this study, we created and characterized alpha cell-specific insulin receptor knockout (alphaIRKO) mice to directly explore the role of insulin signaling in the regulation of glucagon secretion in vivo. Adult male alphaIRKO mice exhibited mild glucose intolerance, hyperglycemia, and hyperglucagonemia in the fed state and enhanced glucagon secretion in response to L-arginine stimulation. Hyperinsulinemic-hypoglycemic clamp studies revealed an enhanced glucagon secretory response and an abnormal norepinephrine response to hypoglycemia in alphaIRKO mice. The mutants also exhibited an age-dependent increase in beta cell mass. Furthermore, siRNA-mediated knockdown of insulin receptor in glucagon-secreting InR1G cells promoted enhanced glucagon secretion and complemented our in vivo findings. Together, these data indicate a significant role for intraislet insulin signaling in the regulation of alpha cell function in both normo- and hypoglycemic conditions.

Ablation of PDK1 in pancreatic β cells induces diabetes as a result of loss of β cell mass

The total mass of islets of Langerhans is reduced in individuals with type 2 diabetes, possibly contributing to the pathogenesis of this condition. Although the regulation of islet mass is complex, recent studies have suggested the importance of a signaling pathway that includes the insulin or insulin-like growth factor–1 receptors, insulin receptor substrate and phosphatidylinositol (PI) 3-kinase. 3-Phosphoinositide–dependent protein kinase 1 (PDK1) is a serine-threonine kinase that mediates signaling downstream of PI 3-kinase. Here we show that mice that lack PDK1 specifically in pancreatic β cells (βPdk1−/− mice) develop progressive hyperglycemia as a result of a loss of islet mass. The mice show reductions in islet density as well as in the number and size of cells. Haploinsufficiency of the gene for the transcription factor Foxo1 resulted in a marked increase in the number, but not the size, of cells and resulted in the restoration of glucose homeostasis in βPdk1−/− mice. These results suggest that PDK1 is important in maintenance of pancreatic cell mass and glucose homeostasis.

Stabilization of β-catenin impacts pancreas growth

A recent study has shown that deletion of β-catenin within the pancreatic epithelium results in a loss of pancreas mass. Here, we show that ectopic stabilization of β-catenin within mouse pancreatic epithelium can have divergent effects on both organ formation and growth. Robust stabilization of β-catenin during early organogenesis drives changes in hedgehog and Fgf10 signaling and induces a loss of Pdx1 expression in early pancreatic progenitor cells. Together, these perturbations in early pancreatic specification culminate in a severe reduction of pancreas mass and postnatal lethality. By contrast, inducing the stabilized form of β-catenin at a later time point in pancreas development causes enhanced proliferation that results in a dramatic increase in pancreas organ size. Taken together, these data suggest a previously unappreciated temporal/spatial role forβ -catenin signaling in the regulation of pancreas organ growth.

Defective insulin secretion in pancreatic β cells lacking type 1 IGF receptor

Defective insulin secretion is a feature of type 2 diabetes that results from inadequate compensatory increase of beta cell mass and impaired glucose-dependent insulin release. beta cell proliferation and secretion are thought to be regulated by signaling through receptor tyrosine kinases. In this regard, we sought to examine the potential proliferative and/or antiapoptotic role of IGFs in beta cells by tissue-specific conditional mutagenesis ablating type 1 IGF receptor (IGF1R) signaling. Unexpectedly, lack of functional IGF1R did not affect beta cell mass, but resulted in age-dependent impairment of glucose tolerance, associated with a decrease of glucose- and arginine-dependent insulin release. These observations reveal a requirement of IGF1R-mediated signaling for insulin secretion.

Mice conditionally lacking the Wolfram gene in pancreatic islet beta cells exhibit diabetes as a result of enhanced endoplasmic reticulum stress and apoptosis

Aims/hypothesisWolfram syndrome is an autosomal recessive disorder characterised by childhood diabetes mellitus, optic atrophy and severe neurodegeneration, resulting in premature death. The aim of this study was to investigate the mechanisms responsible for the phenotype of carbohydrate intolerance and loss of pancreatic beta cells in this disorder.Materials and methodsTo study the role of the Wolfram gene (Wfs1) in beta cells, we developed a mouse model with conditional deletion of Wfs1 in beta cells by crossing floxed Wfs1 exon 8 animals with mice expressing Cre recombinase under the control of a rat insulin promoter (RIP2-Cre). Complementary experiments using RNA interference of Wfs1 expression were performed in mouse insulinoma (MIN6) cell lines (WfsKD).ResultsMale knockout mice (βWfs−/−) began developing variable and progressive glucose intolerance and concomitant insulin deficiency, compared with littermate controls, by 12 weeks of age. Analysis of islets from βWfs−/− mice revealed a reduction in beta cell mass, enhanced apoptosis, elevation of a marker of endoplasmic reticulum stress (immunoglobulin heavy chain-binding protein [BiP]), and dilated endoplasmic reticulum with decreased secretory granules by electron microscopy. WfsKD cell lines had significantly increased apoptosis and elevated expression of the genes encoding BiP and C/EBP-homologous protein (CHOP), two markers of endoplasmic reticulum stress.Conclusions/interpretationThese results indicate that (1) lack of expression of Wfs1 in beta cells was sufficient to result in the diabetes mellitus phenotype; (2) beta cell death occurred by an accelerated process of apoptosis; and (3) lack of Wfs1 was associated with dilated endoplasmic reticulum and increased markers of endoplasmic reticulum stress, which appears to be a significant contributor to the reduction in beta cell survival.

Diabetes recovery by age-dependent conversion of pancreatic δ-cells into insulin producers

Total or near-total loss of insulin-producing β-cells occurs in type 1 diabetes. Restoration of insulin production in type 1 diabetes is thus a major medical challenge. We previously observed in mice in which β-cells are completely ablated that the pancreas reconstitutes new insulin-producing cells in the absence of autoimmunity. The process involves the contribution of islet non-β-cells; specifically, glucagon-producing α-cells begin producing insulin by a process of reprogramming (transdifferentiation) without proliferation. Here we show the influence of age on β-cell reconstitution from heterologous islet cells after near-total β-cell loss in mice. We found that senescence does not alter α-cell plasticity: α-cells can reprogram to produce insulin from puberty through to adulthood, and also in aged individuals, even a long time after β-cell loss. In contrast, before puberty there is no detectable α-cell conversion, although β-cell reconstitution after injury is more efficient, always leading to diabetes recovery. This process occurs through a newly discovered mechanism: the spontaneous en masse reprogramming of somatostatin-producing δ-cells. The juveniles display ‘somatostatin-to-insulin’ δ-cell conversion, involving dedifferentiation, proliferation and re-expression of islet developmental regulators. This juvenile adaptability relies, at least in part, upon the combined action of FoxO1 and downstream effectors. Restoration of insulin producing-cells from non-β-cell origins is thus enabled throughout life via δ- or α-cell spontaneous reprogramming. A landscape with multiple intra-islet cell interconversion events is emerging, offering new perspectives for therapy.

Pancreatic β Cells Require NeuroD to Achieve and Maintain Functional Maturity

NeuroD, a transactivator of the insulin gene, is critical for development of the endocrine pancreas, and NeuroD mutations cause MODY6 in humans. To investigate the role of NeuroD in differentiated beta cells, we generated mice in which neuroD is deleted in insulin-expressing cells. These mice exhibit severe glucose intolerance. Islets lacking NeuroD respond poorly to glucose and display a glucose metabolic profile similar to immature beta cells, featuring increased expression of glycolytic genes and LDHA, elevated basal insulin secretion and O2 consumption, and overexpression of NPY. Moreover, the mutant islets appear to have defective K(ATP) channel-mediated insulin secretion. Unexpectedly, virtually all insulin in the mutant mice is derived from ins2, whereas ins1 expression is almost extinguished. Overall, these results indicate that NeuroD is required for beta cell maturation and demonstrate the importance of NeuroD in the acquisition and maintenance of fully functional glucose-responsive beta cells.