Matrix design for optimal pancreatic ß cells transplantation.

New therapeutic approaches to treat type 1 diabetes mellitus relies on pancreatic islet transplantation. Here, developing immuno-isolation strategies is essential to eliminate the need for systemic immunosuppression after pancreatic islet grafts. A solution is the macro-encapsulation of grafts in semipermeable matrixes with a double function: separating islets from host immune cells and facilitating the diffusion of insulin, glucose, and other metabolites. This study aims to synthesize and characterize different types of gelatin-collagen matrixes to prepare a macro-encapsulation device for pancreatic islets that fulfill these functions. While natural polymers exhibit superior biocompatibility compared to synthetic ones, their mechanical properties are challenging to reproduce. To address this issue, we conducted a comparative analysis between photo-crosslinked gelatin matrixes and chemically crosslinked collagen matrixes. We show that the different crosslinkers and polymerization methods influence the survival and glucose-stimulated insulin production of pancreatic ß cells (INS1) in vitro, as well as the in vitro and in vivo stability of the matrix and the immuno-isolation in vivo. Among the matrixes, the stiff multilayer GelMA matrixes (8.5 kPa), fabricated by digital light processing, were the best suited for pancreatic ß cells macro-encapsulation regarding these parameters. Within the alveoli of this matrix, pancreatic ß cells spontaneously formed aggregates.

A microfluidic platform integrating functional vascularized organoids-on-chip.

The development of vascular networks in microfluidic chips is crucial for the long-term culture of three-dimensional cell aggregates such as spheroids, organoids, tumoroids, or tissue explants. Despite rapid advancement in microvascular network systems and organoid technologies, vascularizing organoids-on-chips remains a challenge in tissue engineering. Most existing microfluidic devices poorly reflect the complexity of in vivo flows and require complex technical set-ups. Considering these constraints, we develop a platform to establish and monitor the formation of endothelial networks around mesenchymal and pancreatic islet spheroids, as well as blood vessel organoids generated from pluripotent stem cells, cultured for up to 30 days on-chip. We show that these networks establish functional connections with the endothelium-rich spheroids and vascular organoids, as they successfully provide intravascular perfusion to these structures. We find that organoid growth, maturation, and function are enhanced when cultured on-chip using our vascularization method. This microphysiological system represents a viable organ-on-chip model to vascularize diverse biological 3D tissues and sets the stage to establish organoid perfusions using advanced microfluidics.

Insight into continuous glucose monitoring: from medical basics to commercialized devices.

According to the latest statistics, more than 537 million people around the world struggle with diabetes and its adverse consequences. As well as acute risks of hypo- or hyper- glycemia, long-term vascular complications may occur, including coronary heart disease or stroke, as well as diabetic nephropathy leading to end-stage disease, neuropathy or retinopathy. Therefore, there is an urgent need to improve diabetes management to reduce the risk of complications but also to improve patient's quality life. The impact of continuous glucose monitoring (CGM) is well recognized, in this regard. The current review aims at introducing the basic principles of glucose sensing, including electrochemical and optical detection, summarizing CGM technology, its requirements, advantages, and disadvantages. The role of CGM systems in the clinical diagnostics/personal testing, difficulties in their utilization, and recommendations are also discussed. In the end, challenges and prospects in future CGM systems are discussed and non-invasive, wearable glucose biosensors are introduced. Though the scope of this review is CGMs and provides information about medical issues and analytical principles, consideration of broader use will be critical in future if the right systems are to be selected for effective diabetes management.

[Generating pancreatic islets organoids: Langerhanoids]. / Les Langerhanoïdes, des organoïdes d'îlots pancréatiques.

The extension of islet transplantation to a wider number of Type 1 diabetic patients is compromised by the scarcity of donors, the reduced ex vivo survival of pancreatic islets and the use of immunosuppressive treatments. Islets of Langerhans isolated from brain-dead donors are currently the only cell source for transplantation. Thus, it is crucial to find an alternative and an abundant source of functional insulin secreting cells not only for clinical use but also for the development of research dedicated to the screening of drugs and to the development of new therapeutic targets. Several groups around the world, including ours, develop 3D culture models as Langerhanoids that closely mimick human pancreatic islets physiology. In this review, we describe recent advances to mimic the pancreatic niche (extracellular matrix, vascularization, microfluidics) allowing better functionality of Langerhanoids.

TITLE: Les Langerhanoïdes, des organoïdes d'îlots pancréatiques. ABSTRACT: Les îlots de Langerhans isolés de donneurs en état de mort encéphalique constituent actuellement la seule source de cellules pour la transplantation de patients atteints de diabète de type 1. Cette approche thérapeutique reste cependant compromise par la rareté des donneurs et par certains aspects techniques. L'utilisation de sources alternatives de cellules productrices d'insuline est donc un enjeu tant thérapeutique que pour la recherche pharmacologique. Plusieurs équipes dans le monde, dont la nôtre, développent des modèles de culture cellulaire en 3D, les Langerhanoïdes, qui sont physiologiquement proches des îlots pancréatiques humains. Dans cette revue, nous décrivons les récentes avancées mimant la niche pancréatique (matrice extracellulaire, vascularisation, microfluidique), permettant ainsi d'accroître la fonctionnalité de ces Langerhanoïdes.

Microfluidic device integrating a network of hyper-elastic valves for automated glucose stimulation and insulin secretion collection from a single pancreatic islet.

Advances in microphysiological systems have prompted the need for robust and reliable cell culture devices. While microfluidic technology has made significant progress, devices often lack user-friendliness and are not designed to be industrialized on a large scale. Pancreatic islets are often being studied using microfluidic platforms in which the monitoring of fluxes is generally very limited, especially because the integration of valves to direct the flow is difficult to achieve. Considering these constraints, we present a thermoplastic manufactured microfluidic chip with an automated control of fluxes for the stimulation and secretion collection of pancreatic islet. The islet was directed toward precise locations through passive hydrodynamic trapping and both dynamic glucose stimulation and insulin harvesting were done automatically via a network of large deformation valves, directing the reagents and the pancreatic islet toward different pathways. This device we developed enables monitoring of insulin secretion from a single islet and can be adapted for the study of a wide variety of biological tissues and secretomes.

Reduced reticulum-mitochondria Ca2+ transfer is an early and reversible trigger of mitochondrial dysfunctions in diabetic cardiomyopathy.

Type 2 diabetic cardiomyopathy features Ca2+ signaling abnormalities, notably an altered mitochondrial Ca2+ handling. We here aimed to study if it might be due to a dysregulation of either the whole Ca2+ homeostasis, the reticulum-mitochondrial Ca2+ coupling, and/or the mitochondrial Ca2+ entry through the uniporter. Following a 16-week high-fat high-sucrose diet (HFHSD), mice developed cardiac insulin resistance, fibrosis, hypertrophy, lipid accumulation, and diastolic dysfunction when compared to standard diet. Ultrastructural and proteomic analyses of cardiac reticulum-mitochondria interface revealed tighter interactions not compatible with Ca2+ transport in HFHSD cardiomyocytes. Intramyocardial adenoviral injections of Ca2+ sensors were performed to measure Ca2+ fluxes in freshly isolated adult cardiomyocytes and to analyze the direct effects of in vivo type 2 diabetes on cardiomyocyte function. HFHSD resulted in a decreased IP3R-VDAC interaction and a reduced IP3-stimulated Ca2+ transfer to mitochondria, with no changes in reticular Ca2+ level, cytosolic Ca2+ transients, and mitochondrial Ca2+ uniporter function. Disruption of organelle Ca2+ exchange was associated with decreased mitochondrial bioenergetics and reduced cell contraction, which was rescued by an adenovirus-mediated expression of a reticulum-mitochondria linker. An 8-week diet reversal was able to restore cardiac insulin signaling, Ca2+ transfer, and cardiac function in HFHSD mice. Therefore, our study demonstrates that the reticulum-mitochondria Ca2+ miscoupling may play an early and reversible role in the development of diabetic cardiomyopathy by disrupting primarily the mitochondrial bioenergetics. A diet reversal, by counteracting the MAM-induced mitochondrial Ca2+ dysfunction, might contribute to restore normal cardiac function and prevent the exacerbation of diabetic cardiomyopathy.

Detergent-Free Decellularization of the Human Pancreas for Soluble Extracellular Matrix (ECM) Production.

Islet transplantation (ITx) has the potential to become the standard of care in beta cell replacement medicine but its results remain inferior to those obtained with whole pancreas transplantation. The protocols currently used for human islet isolation are under scrutiny because they are based on the enzymatic digestion of the organ, whereby the pancreas is demolished, its connections to the body are lost and islets are irreversibly damaged. Islet damage is characterized by critical factors such as the destruction of the extracellular matrix (ECM), which represents the 3D framework of the islet niche and whose loss is incompatible with islet euphysiology. Researchers are proposing the use of ECM-based scaffolds derived from the mammalian pancreas to address this problem and ultimately improve islet viability, function, and lifespan. Currently available methods to obtain such scaffolds are harsh because they are largely detergent based. Thus, we propose a new, detergent-free method that creates less ECM damage and can preserve critical components of pancreatic ECM. The results show that the newly developed decellularization protocol allowed the achievement of complete DNA clearance while the ECM components were retained. The ECM obtained was tested for cytotoxicity and encapsulated with human pancreatic islets which showed a positive cellular behavior with insulin secretion when stimulated with glucose challenge. Collectively, we propose a new method for the decellularization of the human pancreas without the use of conventional ionic and non-ionic chemical detergents. This protocol and the ECM obtained with it could be of use for both in vitro and in vivo applications.

Improved human islets' viability and functionality with mesenchymal stem cells and arg-gly-asp tripeptides supplementation of alginate micro-encapsulated islets in vitro.

INTRODUCTION: The extension of islet transplantation to a wider number of type 1 diabetes patients is compromised by severe adverse events related to the immunosuppressant therapy required for allogenic islet transplantation. In this context, microencapsulation offers the prospects of immunosuppressive-free therapy by physically isolating islets from the immune system. However, current biomaterials need to be optimized to: improve biocompatibility, guaranty the maintenance of graft viability and functionality, and prevent fibrosis overgrowth around the capsule in vivo. Accumulating evidence suggest that mesenchymal stem cells (MSCs) and anchor points consisting of tripeptides arg-gly-asp (RGD) have cytoprotective effects on pancreatic islets. Here, we investigated the effect of supplementing reference M-rich alginate microcapsules with MSCs and RGD-G rich alginate on bioprocessing as well as on human pancreatic islets viability and functionality. METHODS: We characterized the microcapsules components, and then for the new microcapsule composite product: we analyzed the empty capsules biocompatibility and then investigated the benefits of MSCs and RGD-G rich alginate on viability and functionality on the encapsulated human pancreatic islets in vitro. We performed viability tests by confocal microscopy and glucose stimulated insulin secretion (GSIS) test in vitro to assess the functionality of naked and encapsulated islets. RESULTS: Encapsulation in reference M-rich alginate capsules induced a reduction in viability and functionality compared to naked islets. This side-effect of encapsulation was in part counteracted by the presence of MSCs but the restoration was complete with the combination of both MSCs and the RGD-G rich alginate. CONCLUSIONS: The present findings show that bioprocessing a favorable composite environment inside the M-rich alginate capsule with both MSCs and RGD-G rich alginate improves human islets survival and functionality in vitro.

Human mesenchymal stem cells improve rat islet functionality under cytokine stress with combined upregulation of heme oxygenase-1 and ferritin.

BACKGROUND: Islets of Langerhans transplantation is a promising therapy for type 1 diabetes mellitus, but this technique is compromised by transplantation stresses including inflammation. In other tissues, co-transplantation with mesenchymal stem cells has been shown to reduce damage by improving anti-inflammatory and anti-oxidant defences. Therefore, we probed the protection afforded by bone marrow mesenchymal stem cells to islets under pro-inflammatory cytokine stress. METHODS: In order to evaluate the cytoprotective potential of mesenchymal stem cells on rat islets, co-cultures were exposed to the interleukin-1, tumour necrosis factor α and interferon γ cocktail for 24 h. Islet viability and functionality tests were performed. Reactive oxygen species and malondialdehyde were measured. Expression of stress-inducible genes acting as anti-oxidants and detoxifiers, such as superoxide dismutases 1 and 2, NAD(P)H quinone oxidoreductase 1, heme oxygenase-1 and ferritin H, was compared to non-stressed cells, and the corresponding proteins were measured. Data were analysed by a two-way ANOVA followed by a Holm-Sidak post hoc analysis. RESULTS: Exposure of rat islets to cytokines induces a reduction in islet viability and functionality concomitant with an oxidative status shift with an increase of cytosolic ROS production. Mesenchymal stem cells did not significantly increase rat islet viability under exposure to cytokines but protected islets from the loss of insulin secretion. A drastic reduction of the antioxidant factors heme oxygenase-1 and ferritin H protein levels was observed in islets exposed to the cytokine cocktail with a prevention of this effect by the presence of mesenchymal stem cells. CONCLUSIONS: Our data evidenced that MSCs are able to preserve islet insulin secretion through a modulation of the oxidative imbalance mediated by heme and iron via heme oxygenase-1 and ferritin in a context of cytokine exposure.

New Automatized Method of 3D Multiculture Viability Analysis Based on Confocal Imagery: Application to Islets and Mesenchymal Stem Cells Co-Encapsulation.

Co-encapsulation of pancreatic islets with mesenchymal stem cells in a three-dimensional biomaterial's structure is a promising technique to improve transplantation efficacy and to decrease immunosuppressant therapy. Currently, evaluation of graft quality after co-encapsulation is only based on insulin secretion. Viability measurement in a 3D conformation structure involving two different cell types is complex, mainly performed manually, highly time consuming and examiner dependent. Standardization of encapsulated graft viability analysis before transplantation is a key point for the translation of the method from the bench side to clinical practice. In this study, we developed an automated analysis of islet viability based on confocal pictures processing of cells stained with three probes (Hoechst, propidium iodide, and PKH67). When compared with results obtained manually by different examiners, viability results show a high degree of similarity (under 3% of difference) and a tight correlation (r = 0.894; p < 0.001) between these two techniques. The automated technique offers the advantage of reducing the analysis time by 6 and avoids the examiner's dependent variability factor. Thus, we developed a new efficient tool to standardize the analysis of islet viability in 3D structure involving several cell types, which is a key element for encapsulated graft analysis in clinical practice.

Disruption of Mitochondria-Associated Endoplasmic Reticulum Membrane (MAM) Integrity Contributes to Muscle Insulin Resistance in Mice and Humans.

Modifications of the interactions between endoplasmic reticulum (ER) and mitochondria, defined as mitochondria-associated membranes (MAMs), were recently shown to be involved in the control of hepatic insulin action and glucose homeostasis, but with conflicting results. Whereas skeletal muscle is the primary site of insulin-mediated glucose uptake and the main target for alterations in insulin-resistant states, the relevance of MAM integrity in muscle insulin resistance is unknown. Deciphering the importance of MAMs on muscle insulin signaling could help to clarify this controversy. Here, we show in skeletal muscle of different mice models of obesity and type 2 diabetes (T2D) a marked disruption of ER-mitochondria interactions as an early event preceding mitochondrial dysfunction and insulin resistance. Furthermore, in human myotubes, palmitate-induced insulin resistance is associated with a reduction of structural and functional ER-mitochondria interactions. Importantly, experimental increase of ER-mitochondria contacts in human myotubes prevents palmitate-induced alterations of insulin signaling and action, whereas disruption of MAM integrity alters the action of the hormone. Lastly, we found an association between altered insulin signaling and ER-mitochondria interactions in human myotubes from obese subjects with or without T2D compared with healthy lean subjects. Collectively, our data reveal a new role of MAM integrity in insulin action of skeletal muscle and highlight MAM disruption as an essential subcellular alteration associated with muscle insulin resistance in mice and humans. Therefore, reduced ER-mitochondria coupling could be a common alteration of several insulin-sensitive tissues playing a key role in altered glucose homeostasis in the context of obesity and T2D.

Sulforaphane improves disrupted ER-mitochondria interactions and suppresses exaggerated hepatic glucose production.

AIMS: Exaggerated hepatic glucose production is one of the hallmarks of type 2 diabetes. Sulforaphane (SFN) has been suggested as a new potential anti-diabetic compound. However, the effects of SFN in hepatocytes are yet unclear. Accumulating evidence points to the close structural contacts between the ER and mitochondria, known as mitochondria-associated ER membranes (MAMs), as important hubs for hepatic metabolism. We wanted to investigate whether SFN could affect hepatic glucose production and MAMs. MATERIALS AND METHODS: We used proximity ligation assays, analysis of ER stress markers and glucose production assays in hepatoma cell lines, primary mouse hepatocytes and diabetic animal models. RESULTS: SFN counteracted the increase of glucose production in palmitate-treated mouse hepatocytes. SFN also counteracted palmitate-induced MAM disruptions. Moreover, SFN decreased the ER stress markers CHOP and Grp78. In ob/ob mice, SFN improved glucose tolerance and reduced exaggerated glucose production. In livers of these mice, SFN increased MAM protein content, restored impaired VDAC1-IP3R1 interactions and reduced ER stress markers. In mice on HFHSD, SFN improved glucose tolerance, MAM protein content and ER-mitochondria interactions to a similar extent to that of metformin. CONCLUSIONS: The present findings show that MAMs are severely reduced in animal models of glucose intolerance, which reinforces the role of MAMs as a hub for insulin signaling in the liver. We also show that SFN restores MAMs and improves glucose tolerance by a similar magnitude to that of metformin. These data highlight SFN as a new potential anti-diabetic compound.

Sulforaphane reduces hepatic glucose production and improves glucose control in patients with type 2 diabetes.

A potentially useful approach for drug discovery is to connect gene expression profiles of disease-affected tissues ("disease signatures") to drug signatures, but it remains to be shown whether it can be used to identify clinically relevant treatment options. We analyzed coexpression networks and genetic data to identify a disease signature for type 2 diabetes in liver tissue. By interrogating a library of 3800 drug signatures, we identified sulforaphane as a compound that may reverse the disease signature. Sulforaphane suppressed glucose production from hepatic cells by nuclear translocation of nuclear factor erythroid 2-related factor 2 (NRF2) and decreased expression of key enzymes in gluconeogenesis. Moreover, sulforaphane reversed the disease signature in the livers from diabetic animals and attenuated exaggerated glucose production and glucose intolerance by a magnitude similar to that of metformin. Finally, sulforaphane, provided as concentrated broccoli sprout extract, reduced fasting blood glucose and glycated hemoglobin (HbA1c) in obese patients with dysregulated type 2 diabetes.

Metabolic signaling functions of ER-mitochondria contact sites: role in metabolic diseases.

Beyond the maintenance of cellular homeostasis and the determination of cell fate, ER-mitochondria contact sites, defined as mitochondria-associated membranes (MAM), start to emerge as an important signaling hub that integrates nutrient and hormonal stimuli and adapts cellular metabolism. Here, we summarize the established structural and functional features of MAM and mainly focus on the latest breakthroughs highlighting a crucial role of organelle crosstalk in the control of metabolic homeostasis. Lastly, we discuss recent studies that have revealed the importance of MAM in not only metabolic diseases but also in other pathologies with disrupted metabolism, shedding light on potential common molecular mechanisms and leading hopefully to novel treatment strategies.

Mitochondria-associated endoplasmic reticulum membranes allow adaptation of mitochondrial metabolism to glucose availability in the liver.

Mitochondria-associated endoplasmic reticulum membranes (MAM) play a key role in mitochondrial dynamics and function and in hepatic insulin action. Whereas mitochondria are important regulators of energy metabolism, the nutritional regulation of MAM in the liver and its role in the adaptation of mitochondria physiology to nutrient availability are unknown. In this study, we found that the fasted to postprandial transition reduced the number of endoplasmic reticulum-mitochondria contact points in mouse liver. Screening of potential hormonal/metabolic signals revealed glucose as the main nutritional regulator of hepatic MAM integrity both in vitro and in vivo Glucose reduced organelle interactions through the pentose phosphate-protein phosphatase 2A (PP-PP2A) pathway, induced mitochondria fission, and impaired respiration. Blocking MAM reduction counteracted glucose-induced mitochondrial alterations. Furthermore, disruption of MAM integrity mimicked effects of glucose on mitochondria dynamics and function. This glucose-sensing system is deficient in the liver of insulin-resistant ob/ob and cyclophilin D-KO mice, both characterized by chronic disruption of MAM integrity, mitochondrial fission, and altered mitochondrial respiration. These data indicate that MAM contribute to the hepatic glucose-sensing system, allowing regulation of mitochondria dynamics and function during nutritional transition. Chronic disruption of MAM may participate in hepatic mitochondrial dysfunction associated with insulin resistance.

Study of Endoplasmic Reticulum and Mitochondria Interactions by In Situ Proximity Ligation Assay in Fixed Cells.

Structural interactions between the endoplasmic reticular (ER) and mitochondrial membranes, in domains known as mitochondria-associated membranes (MAM), are crucial hubs for cellular signaling and cell fate. Particularly, these inter-organelle contact sites allow the transfer of calcium from the ER to mitochondria through the voltage-dependent anion channel (VDAC)/glucose-regulated protein 75 (GRP75)/inositol 1,4,5-triphosphate receptor (IP3R) calcium channeling complex. While this subcellular compartment is under intense investigation in both physiological and pathological conditions, no simple and sensitive method exists to quantify the endogenous amount of ER-mitochondria contact in cells. Similarly, MAMs are highly dynamic structures, and there is no suitable approach to follow modifications of ER-mitochondria interactions without protein overexpression. Here, we report an optimized protocol based on the use of an in situ proximity ligation assay to visualize and quantify endogenous ER-mitochondria interactions in fixed cells by using the close proximity between proteins of the outer mitochondrial membrane (VDAC1) and of the ER membrane (IP3R1) at the MAM interface. Similar in situ proximity ligation experiments can also be performed with the GRP75/IP3R1 and cyclophilin D/IP3R1 pairs of antibodies. This assay provides several advantages over other imaging procedures, as it is highly specific, sensitive, and suitable to multiple-condition testing. Therefore, the use of this in situ proximity ligation assay should be helpful to better understand the physiological regulations of ER-mitochondria interactions, as well as their role in pathological contexts.

Disruption of calcium transfer from ER to mitochondria links alterations of mitochondria-associated ER membrane integrity to hepatic insulin resistance.

AIMS/HYPOTHESIS: Mitochondria-associated endoplasmic reticulum membranes (MAMs) are regions of the endoplasmic reticulum (ER) tethered to mitochondria and controlling calcium (Ca(2+)) transfer between both organelles through the complex formed between the voltage-dependent anion channel, glucose-regulated protein 75 and inositol 1,4,5-triphosphate receptor (IP3R). We recently identified cyclophilin D (CYPD) as a new partner of this complex and demonstrated a new role for MAMs in the control of insulin's action in the liver. Here, we report on the mechanisms by which disruption of MAM integrity induces hepatic insulin resistance in CypD (also known as Ppif)-knockout (KO) mice. METHODS: We used either in vitro pharmacological and genetic inhibition of CYPD in HuH7 cells or in vivo loss of CYPD in mice to investigate ER-mitochondria interactions, inter-organelle Ca(2+) exchange, organelle homeostasis and insulin action. RESULTS: Pharmacological and genetic inhibition of CYPD concomitantly reduced ER-mitochondria interactions, inhibited inter-organelle Ca(2+) exchange, induced ER stress and altered insulin signalling in HuH7 cells. In addition, histamine-stimulated Ca(2+) transfer from ER to mitochondria was blunted in isolated hepatocytes of CypD-KO mice and this was associated with an increase in ER calcium store. Interestingly, disruption of inter-organelle Ca(2+) transfer was associated with ER stress, mitochondrial dysfunction, lipid accumulation, activation of c-Jun N-terminal kinase (JNK) and protein kinase C (PKC)ε and insulin resistance in liver of CypD-KO mice. Finally, CYPD-related alterations of insulin signalling were mediated by activation of PKCε rather than JNK in HuH7 cells. CONCLUSIONS/INTERPRETATION: Disruption of IP3R-mediated Ca(2+) signalling in the liver of CypD-KO mice leads to hepatic insulin resistance through disruption of organelle interaction and function, increase in lipid accumulation and activation of PKCε. Modulation of ER-mitochondria Ca(2+) exchange may thus provide an exciting new avenue for treating hepatic insulin resistance.

Mitochondria-associated endoplasmic reticulum membrane (MAM) integrity is required for insulin signaling and is implicated in hepatic insulin resistance.

Mitochondria-associated endoplasmic reticulum (ER) membranes (MAMs) are functional domains between both organelles involved in Ca(2+) exchange, through the voltage-dependent anion channel (VDAC)-1/glucose-regulated protein 75 (Grp75)/inositol 1,4,5-triphosphate receptor (IP3R)-1 complex, and regulating energy metabolism. Whereas mitochondrial dysfunction, ER stress, and altered Ca(2+) homeostasis are associated with altered insulin signaling, the implication of MAM dysfunctions in insulin resistance is unknown. Here we validated an approach based on in situ proximity ligation assay to detect and quantify VDAC1/IP3R1 and Grp75/IP3R1 interactions at the MAM interface. We demonstrated that MAM integrity is required for insulin signaling and that induction of MAM prevented palmitate-induced alterations of insulin signaling in HuH7 cells. Disruption of MAM integrity by genetic or pharmacological inhibition of the mitochondrial MAM protein, cyclophilin D (CypD), altered insulin signaling in mouse and human primary hepatocytes and treatment of CypD knockout mice with metformin improved both insulin sensitivity and MAM integrity. Furthermore, ER-mitochondria interactions are altered in liver of both ob/ob and diet-induced insulin-resistant mice and improved by rosiglitazone treatment in the latter. Finally, increasing organelle contacts by overexpressing CypD enhanced insulin action in primary hepatocytes of diabetic mice. Collectively, our data reveal a new role of MAM integrity in hepatic insulin action and resistance, providing a novel target for the modulation of insulin action.

Depressing mitochondria-reticulum interactions protects cardiomyocytes from lethal hypoxia-reoxygenation injury.

BACKGROUND: Under physiological conditions, Ca(2+) transfer from the endoplasmic reticulum (ER) to mitochondria might occur at least in part at contact points between the 2 organelles and involves the VDAC1/Grp75/IP3R1 complex. Accumulation of Ca(2+) into the mitochondrial matrix may activate the mitochondrial chaperone cyclophilin D (CypD) and trigger permeability transition pore opening, whose role in ischemia/reperfusion injury is well recognized. We questioned here whether the transfer of Ca(2+) from ER to mitochondria might play a role in cardiomyocyte death after hypoxia-reoxygenation. METHODS AND RESULTS: We report that CypD interacts with the VDAC1/Grp75/IP3R1 complex in cardiomyocytes. Genetic or pharmacological inhibition of CypD in both H9c2 cardiomyoblasts and adult cardiomyocytes decreased the Ca(2+) transfer from ER to mitochondria through IP3R under normoxic conditions. During hypoxia-reoxygenation, the interaction between CypD and the IP3R1 Ca(2+) channeling complex increased concomitantly with mitochondrial Ca(2+) content. Inhibition of either CypD, IP3R1, or Grp75 decreased protein interaction within the complex, attenuated mitochondrial Ca(2+) overload, and protected cells from hypoxia-reoxygenation. Genetic or pharmacological inhibition of CypD provided a similar effect in adult mice cardiomyocytes. Disruption of ER-mitochondria interaction via the downregulation of Mfn2 similarly reduced the interaction between CypD and the IP3R1 complex and protected against hypoxia-reoxygenation injury. CONCLUSIONS: Our data (1) point to a new role of CypD at the ER-mitochondria interface and (2) suggest that decreasing ER-mitochondria interaction at reperfusion can protect cardiomyocytes against lethal reperfusion injury through the reduction of mitochondrial Ca(2+) overload via the CypD/VDAC1/Grp75/IP3R1 complex.