Efficient gene delivery and silencing of mouse and human pancreatic islets

<p>Abstract</p> <p>Background</p> <p>In view of the importance of beta cells in glucose homeostasis and the profound repercussions of beta cell pathology on human health, the acquisition of tools to study pancreatic islet function is essential for the design of alternative novel therapies for diabetes. One promising approach toward this goal involves the modification of gene expression profile of beta cells.</p> <p>Results</p> <p>This study describes a new method of gene and siRNA delivery into human pancreatic islets by microporation technology. We demonstrated that mild islet distention with accutase greatly enhanced the transfection efficiency without compromising in vitro function (secretion, apoptosis and viability). As an example, the recently identified gene involved in type 2 diabetes, ZnT8, can be over-expressed or silenced by RNA interference using this technology. Microporation can also be used on rodent islets.</p> <p>Conclusions</p> <p>Taken together, our results demonstrate that microporation technology can be used to modify gene expression in whole rodent and human islets without altering their in vitro function and will be key to the elucidation of the factors responsible for proper islet function.</p

In vivo expression and functional characterization of the zinc transporter ZnT8 in glucose-induced insulin secretion.

International audienceInsulin-secreting pancreatic beta cells are exceptionally rich in zinc. In these cells, zinc is required for zinc-insulin crystallization within secretory vesicles. Secreted zinc has also been proposed to be a paracrine and autocrine modulator of glucagon and insulin secretion in pancreatic alpha and beta cells, respectively. However, little is known about the molecular mechanisms underlying zinc accumulation in insulin-containing vesicles. We previously identified a pancreas-specific zinc transporter, ZnT-8, which colocalized with insulin in cultured beta cells. In this paper we studied its localization in human pancreatic islet cells, and its effect on cellular zinc content and insulin secretion. In human pancreatic islet cells, ZnT-8 was exclusively expressed in insulin-producing beta cells, and colocalized with insulin in these cells. ZnT-8 overexpression stimulated zinc accumulation and increased total intracellular zinc in insulin-secreting INS-1E cells. Furthermore, ZnT-8-overexpressing cells display enhanced glucose-stimulated insulin secretion compared with control cells, only for a high glucose challenge, i.e. >10 mM glucose. Altogether, these data strongly suggest that the zinc transporter ZnT-8 is a key protein for both zinc accumulation and regulation of insulin secretion in pancreatic beta cells

Recherche des gènes impliqués dans les phénomènes de prolifération/dédifférenciation des cellules b pancréatiques humaines (intérêt en thérapie cellulaire)

Le diabète de type 1, caractérisé par la destruction auto-immune des cellules b insulino-sécrétrices, est la forme la plus sévère de diabète. La capacité du pancréas à générer des cellules progénitrices a donné naissance à de nouvelles pistes de recherche sur la thérapie cellulaire du diabète de type 1. Nous avons montré que la prolifération des cellules b sur matrice extra-cellulaire en présence d'HGF entraînait une perte d'expression de marqueurs de différenciation tels que l'insuline et son facteur de transcription PDX-1/IPF-1. Après expansion (6 jours), nous avons essayé de ré-exprimer ces marqueurs par l'intermédiaire de composés connus pour leurs effets sur la prolifération et/ou la différenciation. Le butyrate de sodium nous a permis de réinduire le PDX-1/IPF-1 et l'insuline mais aussi de provoquer la sécrétion de gastrine, un facteur intervenant dans la néogenèse pancréatique. D'autres composés comme le TGF b, le calcitriol, le GLP-1 et l'activine A ont aussi eu des effets positifs sur la ré-expression de certains gènes. Ces premiers résultats indiquent qu'il est possible de réinduire une différenciation cellulaire après expansion de cellules b. Nous avons ensuite essayé d'identifier les gènes modifiés durant la culture à long-terme de cellules b purifiées par FACS. L'analyse de trois pancréas différents sur puces à ADN "maison" spécifiques du pancréas a mis en évidence que sur les 218 gènes étudiés, 49 étaient sous-exprimés (marqueurs de différenciation endocrine) et 76 surexprimés (marqueurs de cellules souches et de cellules exocrines). La transdifférenciation des cellules b montraient une surexpression de PBX-1, une protéine formant un hétérodimère avec PDX-1, et qui module l'activité transcriptionnelle de ce dernier. La neurogénine 3, un facteur clé dans la formation des cellules endocrines était lui aussi surexprimé. Ces résultats permettent d'obtenir une carte précise des gènes impliqués dans la prolifération des cellules b.Type 1 diabetes, caracterized by b cell destruction, is the most severe form of diabetes. The pancreatic capacity to generate progenitor cells have given new development in type 1 diabetes cell therapy. In vitro studies of beta cell proliferation on extracellular matrices plus growth factors (HGF) have highlighted a possible cell expansion technique which was however accompanied with loss of insulin secretion. Herein we showed that human islet cell proliferation was marked by a decreased expression of specific differentiation markers, particularly insulin and his transcription factor IPF-1. After a 6 day expansion period, we tried to reexpress the beta cell differentiation markers with compounds known for their differentiation and/or insulin-secreting properties. Sodium butyrate was a potent factor of IPF-1 and insulin, it also clearly induced secretion of gastrin, a known neogenic factor. Other compounds, namely TGF-b, calcitriol, GLP-1 and Activin A, efficiently enhanced the glucose sensor machinery. Our results indicate that specific beta cell gene expression may be induced after expansion and dedifferentiation. In a second time, we tried to identify genes modulated during long term culture of FACS purified b cells. Analysis of 3 differents pancreata were performed on "in-house" pancreas specific microarrays consisting of 218 genes. The expression of 49 of them was down-regulated (markers of endocrine differentiation), while 76 were induced by cell expansion. Their pattern argues for the transdifferentiation of beta cells into undifferentiated cells that overexpress both PBX1, a protein which can bind as a heterodimer with PDX1 and could switch the nature of the its transcriptional activity, and neurogenin 3, a key factor for the generation of endocrine islet cells. Our findings demonstrate that microarray-based technology provides a powerful tool for identifying genes involved in the regulation of pancreatic beta cell growth.LILLE2-BU Santé-Recherche (593502101) / SudocSudocFranceF

Engineered-inhaled particles: Influence of carbohydrates excipients nature on powder properties and behavior

Pulmonary drug administration has long been used for local or systemic treatment due to several advantages. Dry powder inhalers emerge as the most promising due to efficiency, ecologic, and drug stability concerns. Coarse lactose-carrier is still the gold standard when inhalation powders are developed. Despite some efforts to produce new types of powders, the lung drug deposition is still poorly controlled, which will ultimately impact therapeutic effectiveness. In this study, we developed “engineered-inhalation powders” using the spray-drying technique. Multiple carbohydrates excipients were binary mixed and combined with two active pharmaceutical ingredients for asthma therapy (budesonide and formoterol). Particle morphology, from spherical to deflated shapes, was characterized by the number and the depth of dimples measured from SEM images. We define a new characteristic deflation ratio ξ as the product between the number of dimples and their depth. Six different powders having opposite morphologies have been selected and we have demonstrated a linear correlation between the fine particle fraction and the deflation ratio of produced powders. Overall, we showed first that the morphology of inhalable powder can be finely tuned by spray-drying technique when excipients varied. Secondly, we developed stable inhalation powders that simultaneously induced high fine particle fractions (>40%) for two drugs due to their deflated surface. The stability has been evaluated for up to 2 months at room temperature.AEROPER

PPARγ-dependent and -independent effects of Rosiglitazone on lipotoxic human pancreatic islets

International audienceWe explored the in vitro effects of Rosiglitazone (RZG), a PPARgamma agonist, on human pancreatic islet dysfunctions induced by chronic free fatty acid exposure. We demonstrated that RZG beneficial effects on insulin secretion and apoptosis did not imply PDX-1 or insulin gene modulation. It rather involved, through a PPARgamma-dependent mechanism, a reduction of iNOS overexpressed in lipotoxic islets. This reduction likely led to the restoration of ATP level and insulin secretion as well as the decrease in apoptosis. More interestingly, we also demonstrated that RZG beneficial effects involved PPARgamma-independent mechanisms. RZG treatment led to a limitation of oxidative stress exemplified by an increase of GPx and SOD expression. It also increased UCP2 expression that seemed to display antioxidant action in this model. Thus, RZG did not appear to exert a direct action on insulin expression but rather an indirect action on insulin secretion and apoptosis, through PPARgamma-dependent and -independent mechanisms, via regulation of nitrogen and oxygen reactive species injury

Engineered-inhaled particles: influence of carbohydrates excipients nature on powder properties and behavior

peer reviewedPulmonary drug administration has long been used for local or systemic treatment due to several advantages. Dry powder inhalers (DPI) emerge as the most promising due to efficiency, ecologic, and drug stability concerns. Coarse lactose-carrier is still the gold standard when inhalation powders are developed. Despite some efforts to produce new types of powders, the lung drug deposition is still poorly controlled which will ultimately impact therapeutic effectiveness. The goal of this study is to develop an inhaled powder leading to a deep and uniform deposition of 2 different active pharmaceutical ingredients (API). We developed “engineered-inhalation powders” using the spray-drying technique. Multiple carbohydrates excipients were binary mixed and combined with two active pharmaceutical ingredients for asthma therapy (budesonide and formoterol). Particle morphology was characterized by the number and the depth of dimples, measured from SEM images. We define a new characteristic deflation ratio ξ as the product between the number of dimples and their depth. Six different powders having opposite morphologies have been selected and we have demonstrated a linear correlation between the fine particle fraction (FPF) and the deflation ratio of produced powders.AEROPERF - Win2Wa

5′-AZA induces Ngn3 expression and endocrine differentiation in the PANC-1 human ductal cell line

International audienceNeurogenin 3 is necessary for endocrine cell development in the embryonic pancreas and has been shown to induce transdifferentiation duct cells from adult pancreas toward a neuro-endocrine phenotype. Here we discovered that the demethylating agent 5'-Azadeoxycytidine (AZA) induced Ngn3 expression and endocrine differentiation from the PANC-1 human ductal cell line. The expression of markers specific to mature islet cells, i.e., glucagon and somatostatin, was also observed. In addition, we demonstrated that growth factors (betacellulin and soluble factors released during pancreas embryogenesis) increased the level of maturation. Our studies revealed that the PANC-1 model system may provide a basis for elucidating the ductal/endocrine differentiation