β-Cell Replacement Strategies: The Increasing Need for a “β-Cell Dogma”

Type 1 diabetes is an auto-immune disease resulting in the loss of pancreatic β-cells and, consequently, in chronic hyperglycemia. Insulin supplementation allows diabetic patients to control their glycaemia quite efficiently, but treated patients still display an overall shortened life expectancy and an altered quality of life as compared to their healthy counterparts. In this context and due to the ever increasing number of diabetics, establishing alternative therapies has become a crucial research goal. Most current efforts therefore aim at generating fully functional insulin-secreting β-like cells using multiple approaches. In this review, we screened the literature published since 2011 and inventoried the selected markers used to characterize insulin-secreting cells generated by in vitro differentiation of stem/precursor cells or by means of in vivo transdifferentiation. By listing these features, we noted important discrepancies when comparing the different approaches for the initial characterization of insulin-producing cells as true β-cells. Considering the recent advances achieved in this field of research, the necessity to establish strict guidelines has become a subject of crucial importance, especially should one contemplate the next step, which is the transplantation of in vitro or ex vivo generated insulin-secreting cells in type 1 diabetic patients

GABA signaling stimulates α-cell-mediated β-like cell neogenesis

Diabetes is a chronic and progressing disease, the number of patients increasing exponentially, especially in industrialized countries. Regenerating lost insulin-producing cells would represent a promising therapeutic alternative for most diabetic patients. To this end, using the mouse as a model, we reported that GABA, a food supplement, could induce insulin-producing beta-like cell neogenesis offering an attractive and innovative approach for diabetes therapeutics

Genetic influence in liver steatosis prevalence and proatherothrombotic/inflammatory profile in familial combined hyperlipoproteinemia

13nononemixedFrancesca Santilli;Patrizia Blardi;Francesca Scarpini;Angela Acciavatti;Linda Ceccatelli;Antonio Magliocca;Tiziana Avolio;Monica Bocchia;Carlo Scapellato;Walter Renato Gioffrè;Alberto Auteri;Silvia Cristina Ferracane;Luca PuccettiFrancesca, Santilli; Blardi, Patrizia; Francesca, Scarpini; Angela, Acciavatti; Linda, Ceccatelli; Antonio, Magliocca; Tiziana, Avolio; Bocchia, Monica; Carlo, Scapellato; Walter Renato, Gioffrè; Alberto, Auteri; Silvia Cristina, Ferracane; Puccetti, Luc

<i>Neurog3</i> misexpression unravels mouse pancreatic ductal cell plasticity

<div><p>In the context of type 1 diabetes research and the development of insulin-producing β-cell replacement strategies, whether pancreatic ductal cells retain their developmental capability to adopt an endocrine cell identity remains debated, most likely due to the diversity of models employed to induce pancreatic regeneration. In this work, rather than injuring the pancreas, we developed a mouse model allowing the inducible misexpression of the proendocrine gene <i>Neurog3</i> in ductal cells <i>in vivo</i>. These animals developed a progressive islet hypertrophy attributed to a proportional increase in all endocrine cell populations. Lineage tracing experiments indicated a continuous neo-generation of endocrine cells exhibiting a ductal ontogeny. Interestingly, the resulting supplementary β-like cells were found to be functional. Based on these findings, we suggest that ductal cells could represent a renewable source of new β-like cells and that strategies aiming at controlling the expression of <i>Neurog3</i>, or of its molecular targets/co-factors, may pave new avenues for the improved treatments of diabetes.</p></div

Analysis of the <i>Neurog3</i> misexpression efficiency in HNFN3OE mice following short-term tamoxifen induction.

<p>(<b>A-B</b>) β-galactosidase activity assessment in the pancreata of HNFN3OE animals treated with vehicle (<b>A</b>) or Tam (<b>B</b>) for 2 weeks. A clear activity is noted solely in ductal cells. (<b>C</b>) Quantitative analysis of <i>Neurog3</i> transcript levels by qPCR (n = 6 animals for each condition) outlining a 2.5-fold increase in the pancreata of Tam-treated HNFN3OE animals compared to controls. Statistics were performed using the Mann-Whitney test (<b>D-E</b>) By means of immunohistochemical analyses using antibodies raised against Neurog3, Neurog3-expressing cells are detected within the ductal epithelium of HNFN3OE animals treated with Tam for only 2 weeks (<b>E</b>), whereas Neurog3⁺ cells cannot be detected in their vehicle-treated counterparts (<b>D</b>). For clarity, when required, the ductal lumen is outlined with yellow lines and islets with red lines.</p

Lineage tracing experiments unravel the conversion of ductal cells into endocrine cells upon the sole <i>Neurog3</i> misexpression.

<p>(<b>A</b>) Taking advantage of the β-galactosidase tracer, we monitored the fate of the ductal cells ectopically expressing <i>Neurog3</i>. X-Gal staining reveals β-galactosidase-positive cells (previously ductal cells) within the islet of Langerhans (outlined with red lines) of Tam-treated HNFN3OE mice. (<b>B</b>) Quantitative RT-PCR analyses confirm the presence of <i>β-galactosidase</i> mRNA in the transcriptome of islets isolated from Tam-treated animals (n = 6 animals for each condition). Statistics were performed using the Mann-Whitney test (<b>C-D</b>) Immunohistochemical analyses combining β-galactosidase and insulin detection. While control pancreata are negative for β-galactosidase (<b>C</b>), their Tam-treated counterparts display cells positive for both insulin and β-galactosidase (<b>D</b>), indicating duct-to-endocrine cell conversion. Note that the apparent staining in the exocrine tissue is artefactual and caused by the antibody used. (<b>E-F</b>) Accordingly, several glucagon⁺ (<b>E</b>) or somatostatin⁺ (<b>F</b>) cells are also found labeled with the ductal cell tracer, β-galactosidase.</p

Phenotypical analyses of islet cells from Tam-treated HNFN3OE pancreata.

<p>Representative pictures of immunohistochemical analyses performed on pancreas sections from untreated (<b>A, C, E, G, I, K</b>) and age-matched Tam-treated HNFN3OE mice (<b>B, D, F, H, J, L</b>) using the indicated antibody combinations. All insulin⁺ cells (preexisting and neogenerated) express the <i>bona fide</i> β-cell markers Nkx6.1 (<b>A-B</b>), NeuroD1 (<b>C-D</b>), Pdx1 (<b>E-F</b>), Rfx6 (<b>G-H</b>), Glut2 (<b>I-J</b>), and PC1/3 (<b>K-L</b>).</p

<i>Neurog3</i> misexpression in insulin⁺ cells does not alter β-cell function.

<p>(<b>A</b>) A combination of immunohistochemical detection and Salmon-Gal staining was used to assess β-galactosidase activity in InsCre::Neurog3OE pancreata. 80% of insulin-producing cells appeared positive for β-galactosidase. For clarity, Salmon-Gal staining was converted to a green labeling in the photographs. (<b>B</b>) Intraperitoneal glucose tolerance tests of adult InsCre::Neurog3OE animals (5-month old, n = 5) do not show any difference in glucose response comparing transgenic animals and their age-matched transgene-negative littermates (n = 4), indicating that maintained <i>Neurog3</i> misexpression in β-cells does not impair their ability to secrete insulin in response to glucose stimulation. Statistics were performed using the Mann-Whitney test.</p