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

Pharmacological approach to understanding the control of insulin secretion in human islets.

Aims: To understand better the control of insulin secretion by human β cells and to identify similarities to and differences from rodent models. Methods: Dynamic insulin secretion was measured in perifused human islets treated with pharmacological agents of known modes of action. Results: Glucokinase activation (Ro28-1675) lowered the glucose threshold for stimulation of insulin secretion to 1 mmol/L (G1), augmented the response to G3-G5 but not to G8-G15, whereas tolbutamide remained active in G20, which indicates that not all KATP channels were closed by high glucose concentrations. An almost 2-fold greater response to G15 than to supramaximal tolbutamide in G3 or to KCl+diazoxide in G15 vs G3 quantified the contribution of metabolic amplification to insulin secretion. Both disruption (latrunculin-B) and stabilization (jasplakinolide) of microfilaments augmented insulin secretion without affecting metabolic amplification. Tolbutamide-induced insulin secretion was consistently greater in G10 than G3, with a threshold at 1 and maximum at 10 μmol/L tolbutamide in G10, vs 10 and 25 μmol/L in G3. Sulphonylurea effects were thus clearly glucose-dependent. Insulin secretion was also increased by inhibiting K channels other than KATP channels: Kv or BK channels (tetraethylammonium), TASK-1 channels (ML-365) and SK4 channels (TRAM-34). Opening KATP channels with diazoxide inhibited glucose-induced insulin secretion with half maximum inhibitory concentrations of 9.6 and 24 μmol/L at G7 and G15. Blockade of L-type Ca channels (nimodipine) abolished insulin secretion, whereas a blocker of T-type Ca channels (NNC-55-0396) was ineffective at specific concentrations. Blockade of Na channels (tetrodotoxin) did not affect glucose-induced insulin secretion. Conclusions: In addition to sharing a KATP channel-dependent triggering pathway and a metabolic amplifying pathway, human and rodent β cells were found to display more similarities than differences in the control of insulin secretion

mTORC1 and mTORC2 regulate insulin secretion through Akt in INS-1 cells

International audienceRegulated associated protein of mTOR (Raptor) and rapamycin-insensitive companion of mTOR (rictor) are two proteins that delineate two different mTOR complexes, mTORC1 and mTORC2 respectively. Recent studies demonstrated the role of rictor in the development and function of β-cells. mTORC1 has long been known to impact β-cell function and development. However, most of the studies evaluating its role used either drug treatment (i.e. rapamycin) or modification of expression of proteins known to modulate its activity, and the direct role of raptor in insulin secretion is unclear. In this study, using siRNA, we investigated the role of raptor and rictor in insulin secretion and production in INS-1 cells and the possible cross talk between their respective complexes, mTORC1 and mTORC2. Reduced expression of raptor is associated with increased glucose-stimulated insulin secretion and intracellular insulin content. Downregulation of rictor expression leads to impaired insulin secretion without affecting insulin content and is able to correct the increased insulin secretion mediated by raptor siRNA. Using dominant-negative or constitutively active forms of Akt, we demonstrate that the effect of both raptor and rictor is mediated through alteration of Akt signaling. Our finding shed new light on the mechanism of control of insulin secretion and production by the mTOR, and they provide evidence for antagonistic effect of raptor and rictor on insulin secretion in response to glucose by modulating the activity of Akt, whereas only raptor is able to control insulin biosynthesis

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

Regulation and functional effects of ZNT8 in human pancreatic islets

International audienceZinc ions are essential for the formation of insulin crystals in pancreatic β cells, thereby contributing to packaging efficiency of stored insulin. Zinc fluxes are regulated through the SLC30A (zinc transporter, ZNT) family. Here, we investigated the effect of metabolic stress associated with the prediabetic state (zinc depletion, glucotoxicity, and lipotoxicity) on ZNT expression and human pancreatic islet function. Both zinc depletion and lipotoxicity (but not glucotoxicity) downregulated ZNT8 ( SLC30A8 ) expression and altered the glucose-stimulated insulin secretion index (GSIS). ZNT8 overexpression in human islets protected them from the decrease in GSIS induced by tetrakis-(2-pyridylmethyl) ethylenediamine and palmitate but not from cell death. In addition, zinc supplementation decreased palmitate-induced human islet cell death without restoring GSIS. Altogether, we showed that ZNT8 expression responds to variation in zinc and lipid levels in human β cells, with repercussions on insulin secretion. Prospects for increasing ZNT8 expression and/or activity may prove beneficial in type 2 diabetes in humans

Islets for research: nothing is perfect, but we can do better

In December 2018, Diabetes and Diabetologia began requiring authors of papers reporting data obtained from studies on human islets to report critical characteristics of the human islets used for research. The islet community was asked to provide feedback on it. Here is the contribution by the European Consortium for Islet Transplantation