Mitochondria-associated ER Membranes Integrity Research Articles

Particulate matter induced cognitive impairments via endoplasmic reticulum stress-mediated damage to mitochondria-associated endoplasmic reticulum membranes in immature rats

Particulate matter (PM) exposure has been increasingly recognized as detrimental to cognitive function and is associated with neurodevelopmental disorders. Mitochondria-associated endoplasmic reticulum membranes (MAMs) form an integrated interface between mitochondria and the endoplasmic reticulum (ER), facilitating crucial cellular functions. Prolonged ER stress (ERS) is implicated in various pathological states in the nervous system. MAMs and ERS may play vital roles in adverse effects of early-life PM exposure on cognitive abilities. This study investigated whether ERS plays a role in PM-induced MAMs dysfunction, leading to neuronal damage and cognitive impairments in early postnatal rats. Using a rat model with PM exposure concentrations of 2 and 10 mg/kg from postnatal Day 3 (PND3) to PND28, we observed that PM exposure resulted in anxiety-like behavior and impaired spatial working memory. The protein levels of ERS markers, including GRP78 and CHOP, were significantly increased in response to PM exposure. Western blot, transmission electron microscopy (TEM), and immunofluorescence analyses revealed decreased MAMs-related proteins and disrupted MAM structure and function caused by PM exposure. Administration of the ERS inhibitor 4-phenylbutyric acid (4-PBA) ameliorated these effects, restoring MAMs integrity and improving cognitive deficits. These findings highlighted the key role of ERS-MAMs dysfunction in PM-induced neurotoxicity and cognitive impairments, providing a new perspective and strategy for the prevention of cognitive deficits in early age with PM exposure.

Hesperidin alleviates terbuthylazine-induced ferroptosis via maintenance of mitochondria-associated endoplasmic reticulum membrane integrity in chicken hepatocytes

Terbuthylazine (TBA) is a common triazine herbicide used in agricultural production, which causes toxic damage in multiple tissues. Hesperidin (HSP) is a flavonoid derivative that has anti-inflammatory, antioxidant and cytoprotective effects, but its role in reducing toxic damage caused by pesticides is still unclear. In this study, we aimed to investigate the toxic effect of TBA exposure on chicken hepatocytes and the therapeutic effect of HSP on the TBA-induced hepatotoxicity. Our results demonstrated that HSP could alleviate TBA exposure-induced endoplasmic reticulum (ER) stress. Interestingly, TBA significantly disrupted the integrity of mitochondria-associated endoplasmic reticulum membrane (MAM), while HSP treatment showed the opposite tendency. In addition, TBA could significantly trigger ferroptosis in liver, and HSP treatment reversed ferroptosis under TBA exposure. These results suggested that HSP could inhibit ER stress and alleviate ferroptosis under TBA exposure via maintaining MAM integrity, which provided a novel strategy to take precautions against TBA toxicity.

Disruption of MAM integrity in mutant FUS oligodendroglial progenitors from hiPSCs

Amyotrophic lateral sclerosis (ALS) is a rapidly progressive and fatal neurodegenerative disorder, characterized by selective loss of motor neurons (MNs). A number of causative genetic mutations underlie the disease, including mutations in the fused in sarcoma (FUS) gene, which can lead to both juvenile and late-onset ALS. Although ALS results from MN death, there is evidence that dysfunctional glial cells, including oligodendroglia, contribute to neurodegeneration. Here, we used human induced pluripotent stem cells (hiPSCs) with a R521H or a P525L mutation in FUS and their isogenic controls to generate oligodendrocyte progenitor cells (OPCs) by inducing SOX10 expression from a TET-On SOX10 cassette. Mutant and control iPSCs differentiated efficiently into OPCs. RNA sequencing identified a myelin sheath-related phenotype in mutant OPCs. Lipidomic studies demonstrated defects in myelin-related lipids, with a reduction of glycerophospholipids in mutant OPCs. Interestingly, FUSR521H OPCs displayed a decrease in the phosphatidylcholine/phosphatidylethanolamine ratio, known to be associated with maintaining membrane integrity. A proximity ligation assay further indicated that mitochondria-associated endoplasmic reticulum membranes (MAM) were diminished in both mutant FUS OPCs. Moreover, both mutant FUS OPCs displayed increased susceptibility to ER stress when exposed to thapsigargin, and exhibited impaired mitochondrial respiration and reduced Ca2+ signaling from ER Ca2+ stores. Taken together, these results demonstrate a pathological role of mutant FUS in OPCs, causing defects in lipid metabolism associated with MAM disruption manifested by impaired mitochondrial metabolism with increased susceptibility to ER stress and with suppressed physiological Ca2+ signaling. As such, further exploration of the role of oligodendrocyte dysfunction in the demise of MNs is crucial and will provide new insights into the complex cellular mechanisms underlying ALS.

Genome-wide kinase-MAM interactome screening reveals the role of CK2A1 in MAM Ca2+ dynamics linked to DEE66

The endoplasmic reticulum (ER) and mitochondria form a unique subcellular compartment called mitochondria-associated ER membranes (MAMs). Disruption of MAMs impairs Ca2+ homeostasis, triggering pleiotropic effects in the neuronal system. Genome-wide kinase-MAM interactome screening identifies casein kinase 2 alpha 1 (CK2A1) as a regulator of composition and Ca2+ transport of MAMs. CK2A1-mediated phosphorylation of PACS2 at Ser207/208/213 facilitates MAM localization of the CK2A1-PACS2-PKD2 complex, regulating PKD2-dependent mitochondrial Ca2+ influx. We further reveal that mutations of PACS2 (E209K and E211K) associated with developmental and epileptic encephalopathy-66 (DEE66) impair MAM integrity through the disturbance of PACS2 phosphorylation at Ser207/208/213. This, in turn, causes the reduction of mitochondrial Ca2+ uptake and the dramatic increase of the cytosolic Ca2+ level, thereby, inducing neurotransmitter release at the axon boutons of glutamatergic neurons. In conclusion, our findings suggest a molecular mechanism that MAM alterations induced by pathological PACS2 mutations modulate Ca2+-dependent neurotransmitter release.

Mitofusin2 Ameliorated Endoplasmic Reticulum Stress and Mitochondrial Reactive Oxygen Species Through Maintaining Mitochondria-Associated Endoplasmic Reticulum Membrane Integrity in Cisplatin-Induced Acute Kidney Injury.

Aims: This study investigated the regulatory effect of Mitofusin2 (Mfn2) on mitochondria-associated endoplasmic reticulum membrane (MAM) integrity and cellular injury in cisplatin-induced acute kidney injury (CP-AKI). Results: CP-AKI mice exhibited decreased expression of Mfn2, increased expression of phosphorylated adenosine monophosphate-activated protein kinase (p-AMPK), abnormal mitochondrial morphology, and reduced MAMs integrity, accompanied by the activation of mitochondrial reactive oxygen species (ROS) and endoplasmic reticulum (ER) stress (inositol-requiring enzyme 1 [IRE1] and PERK pathways). In in vitro studies, CP-induced mitochondrial ROS, ER-stress activation, and increased apoptosis were accompanied by the downregulation of Mfn2 and MAMs integrity reduction in Boston University mouse proximal tubular cells (BUMPT) and human proximal tubular epithelial cells (HK-2). Pretreatment of BUMPT cells with the Mfn2 plasmid partially restored the integrity of MAMs, negatively controlled IRE1 and PERK pathways, and inhibited cell apoptosis. In contrast, ER-stress and MAMs integrity violations were increased after Mfn2 small-interfering RNA (siRNA) treatment in HK-2 cells under CP treatment. Coimmunoprecipitation analysis demonstrated that Mfn2 interacted with PERK and IRE1. Furthermore, the adenosine 5'-monophosphate (AMP)-activated protein kinase (AMPK), acadesine (AICAR), had a similar effect to Mfn2 plasmid in the regulation of ER stress and MAMs. Conversely, the ER-stress inhibitor, 4-phenylbutyric acid (4-PBA), had no effect on the expression of Mfn2 and MAMs integrity. Innovation and Conclusion: This is the first study to explore the association between MAMs, ER stress, and Mfn2 in CP-AKI. Downregulation of Mfn2 expression abolished the MAMs integrity, and induced ER stress, mitochondrial ROS, and tubular cell apoptosis. This suggests that the Mfn2-MAMs pathway is a potential therapeutic target in CP-AKI. Antioxid. Redox Signal. 40, 16-39. The Ethical Registration number of animal experiment in this study was CSU-2022-01-0095.

DsbA-L alleviates tubular injury in diabetic nephropathy by activating mitophagy through maintenance of MAM integrity.

Mitochondria-associated endoplasmic reticulum membranes (MAMs) regulate ATG14- and Beclin1-mediated mitophagy and play key roles in the development of diabetic nephropathy (DN). DsbA-L is mainly located in MAMs and plays a role in renoprotection, but whether it activates mitophagy by maintaining MAM integrity remains unclear. In the present study, we found that renal tubular damage was further aggravated in diabetic DsbA-L-/- mice compared with diabetic mice and that this damage was accompanied by disrupted MAM integrity and decreased mitophagy. Furthermore, notably decreased expression of ATG14 and Beclin1 in MAMs extracted from the kidneys of diabetic DsbA-L-/- mice was observed. In vitro, overexpression of DsbA-L reversed the disruption of MAM integrity and enhanced mitophagy in HK-2 cells, a human proximal tubular cell line, after exposure to high-glucose (HG) conditions. Additionally, compared with control mice, DsbA-L-/- mice were exhibited down-regulated expression of helicase with zinc finger 2 (HELZ2) in their kidneys according to transcriptome analysis; HELZ2 serves as a cotranscription factor that synergistically functions with PPARα to promote the expression of mitofusin 2 (MFN-2). Treatment of HK-2 cells with MFN-2 siRNA resulted in MAM uncoupling and decreased mitophagy. Moreover, HG notably reduced the expression of HELZ2 and MFN-2 and inhibited mitophagy, and these effects were partially blocked by overexpression of DsbA-L and altered upon cotreatment with HELZ2 siRNA, HELZ2 overexpression or MK886 (PPARα inhibitor) treatment. These data indicate that DsbA-L alleviates diabetic tubular damage by activating mitophagy through maintenance of MAM integrity via the HELZ2/MFN-2 pathway.

Mitochondria-associated endoplasmic reticulum membranes participate mitochondrial dysfunction and endoplasmic reticulum stress caused by copper in duck kidney.

Copper (Cu) can be harmful to host physiology at high levels, although it is still unclear exactly how it causes nephrotoxicity. Mitochondrial dysfunction and endoplasmic reticulum (ER) stress are associated with heavy metal intoxication. Meanwhile, mitochondria and ER are connected via mitochondria-associated ER membranes (MAM). In order to reveal the crosstalk between them, a total of 144 1-day-old Peking ducks were randomly divided into four groups: control (basal diet), 100mg/kg Cu, 200mg/kg Cu, and 400mg/kg Cu groups. Results found that excessive Cu disrupted MAM integrity, reduced the co-localization of IP3R and VDAC1, and significantly changed the MAM-related factors levels (Grp75, Mfn2, IP3R, MCU, PACS2, and VDAC1), leading to MAM dysfunction. We further found that Cu exposure induced mitochondrial dysfunction via decreasing the ATP level and the expression levels of COX4, TOM20, SIRT1, and OPA1 and up-regulating Parkin expression level. Meanwhile, Cu exposure dramatically increased the expression levels of Grp78, CRT, and ATF4, resulting in ER stress. Overall, these findings demonstrated MAM plays the critical role in Cu-induced kidney mitochondrial dysfunction and ER stress, which deepened our understanding of Cu-induced nephrotoxicity.

The mitochondrial associated endoplasmic reticulum membranes: A platform for the pathogenesis of inflammation-mediated metabolic diseases.

Mitochondria‐associated endoplasmic reticulum membranes (MAM) are specialized subcellular compartments that are shaped by endoplasmic reticulum (ER) subdomains placed side by side to the outer membrane of mitochondria (OMM) being connected by tethering proteins in mammalian cells. Studies showed that MAM has multiple physiological functions. These include regulation of lipid synthesis and transport, Ca2+ transport and signaling, mitochondrial dynamics, apoptosis, autophagy, and formation and activation of an inflammasome. However, alterations of MAM integrity lead to deleterious effects due to an increased generation of mitochondrial reactive oxygen species (ROS) via increased Ca2+ transfer from the ER to mitochondria. This, in turn, causes mitochondrial damage and release of mitochondrial components into the cytosol as damage‐associated molecular patterns which rapidly activate MAM‐resident Nod‐like receptor protein‐3 (NLRP3) inflammasome components. This complex induces the release of pro‐inflammatory cytokines that initiate low‐grade chronic inflammation that subsequently causes the development of metabolic diseases. But, the mechanisms of how MAM is involved in the pathogenesis of these diseases are not exhaustively reviewed. Therefore, this review was aimed to highlight the contribution of MAM to a variety of cellular functions and consider its significance pertaining to the pathogenesis of inflammation‐mediated metabolic diseases.

Ultrastructural and proteomic profiling of mitochondria-associated endoplasmic reticulum membranes reveal aging signatures in striated muscle

Aging is a major risk for developing cardiac and skeletal muscle dysfunction, yet the underlying mechanism remains elusive. Here we demonstrated that the mitochondria-associated endoplasmic reticulum membranes (MAMs) in the rat heart and skeletal muscle were disrupted during aging. Using quantitative morphological analysis, we showed that the mitochondria-endoplasmic reticulum contacts (MERCs) were reduced by half over the lifespan with an early onset of accelerated thickening in the clefts. The ultrastructural changes were further validated by proteomic profiling of the MAM fractions. A combination of subcellular fractionation and quantitative mass spectrometry identified 1306 MAM-enriched proteins in both heart and skeletal muscle, with a catalog of proteins dysregulated with aging. Functional mapping of the MAM proteome suggested several aging signatures to be closely associated with the ER-mitochondria crosstalk, including local metabolic rewiring, calcium homeostasis imbalance, and impaired organelle dynamics and autophagy. Moreover, we identified a subset of highly interconnected proteins in an ER-mitochondria organization network, which were consistently down-regulated with aging. These decreased proteins, including VDAC1, SAMM50, MTX1 and MIC60, were considered as potential contributors to the age-related MAM dysfunction. This study highlights the perturbation in MAM integrity during the striated muscle aging process, and provides a framework for understanding aging biology from the perspective of organelle interactions.

Activation of endoplasmic reticulum-mitochondria coupling drives copper-induced autophagy in duck renal tubular epithelial cells

Copper (Cu) as a transition metal can be toxic to public and ecosystem health at high level, but the specific mechanism of Cu-evoked nephrotoxicity remains elusive. Here, we first revealed the crosstalk between mitofusin2 (Mfn2)-dependent mitochondria-associated endoplasmic reticulum membrane (MAM) dynamics and autophagy in duck renal tubular epithelial cells under Cu exposure. Primary duck renal tubular epithelial cells were treated with 100 and 200 μM Cu sulfate for 12 h and exposed to lentivirus to deliver mitofusin2 (Mfn2). We found that excessive Cu disrupted MAM integrity, decreased the mitochondrial calcium level, co-localization of IP3R and VDAC1, the mRNA levels of PACS2, Mfn2, IP3R and MCU, and Mfn2 and VDAC1 protein levels, causing MAM dysfunction. Furthermore, Mfn2 overexpression ameliorated Cu-induced MAM dysfunction, and increased Cu-evoked autophagy in duck renal tubular epithelial cells accompanied with the elevation of autophagosomes number, ROS level, LC3 puncta, Atg5 and LC3B mRNA levels, and Beclin1, Atg14, LC3BII/LC3BI protein levels. Accordingly, our data proved that excessive Cu could trigger MAM dysfunction and autophagy in duck renal tubular epithelial cells, and Cu-induced autophagy could be activated through Mfn2-dependent MAM, providing evidence on the toxicological exploration mechanisms of Cu.

PACS-2 Ameliorates Tubular Injury by Facilitating Endoplasmic Reticulum-Mitochondria Contact and Mitophagy in Diabetic Nephropathy.

Mitochondria-associated endoplasmic reticulum membrane (MAM) may have a role in tubular injury in diabetic nephropathy (DN), but the precise mechanism remains unclear. Here, we demonstrate that the expression of phosphofurin acidic cluster sorting protein 2 (PACS-2), a critical regulator of MAM formation, is significantly decreased in renal tubules of patients with DN, and PACS-2 expression is positively correlated with renal function and negatively correlated with degrees of tubulointerstitial lesions. Conditional deletion of Pacs-2 in proximal tubules (PTs) aggravates albuminuria and tubular injury in a streptozotocin-induced mouse model of diabetes. Mitochondrial fragmentation, MAM disruption, and defective mitophagy accompanied by altered expression of mitochondrial dynamics and mitophagic proteins, including Drp1 and Becn1, are observed in tubules of diabetic mice; these changes are more pronounced in PT-specific Pacs-2 knockout mice. In vitro, overexpression of PACS-2 in HK-2 cells alleviates excessive mitochondrial fission induced by high glucose concentrations through blocking mitochondrial recruitment of DRP1 and subsequently restores MAM integrity and enhances mitophagy. Mechanistically, PACS-2 binds to BECN1 and mediates the relocalization of BECN1 to MAM, where it promotes the formation of mitophagosome. Together, these data highlight an important but previously unrecognized role of PACS-2 in ameliorating tubular injury in DN by facilitating MAM formation and mitophagy.

The Molecular Mechanisms Underlying Mitochondria-Associated Endoplasmic Reticulum Membrane-Induced Insulin Resistance.

Mitochondria and the endoplasmic reticulum (ER) are connected at multiple sites via what are known as mitochondria-associated ER membranes (MAMs). These associations are known to play an important role in maintaining cellular homeostasis. Impaired MAM signaling has wide-ranging effects in many diseases, such as obesity, diabetes, and neurodegenerative disorders. Accumulating evidence has suggested that MAMs influence insulin signaling through different pathways, including those associated with Ca2+ signaling, lipid metabolism, mitochondrial function, ER stress responses, and inflammation. Altered MAM signaling is a common feature of insulin resistance in different tissues, including the liver, muscle, and even the brain. In the liver, MAMs are key glucose-sensing regulators and have been proposed to be a hub for insulin signaling. Impaired MAM integrity has been reported to disrupt hepatic responses to changes in glucose availability during nutritional transition and to induce hepatic insulin resistance. Meanwhile, these effects can be rescued by the reinforcement of MAM interactions. In contrast, several studies have proposed that enhanced ER-mitochondria connections are detrimental to hepatic insulin signaling and can lead to mitochondrial dysfunction. Thus, given these contradictory results, the role played by the MAM in the regulation of hepatic insulin signaling remains elusive. Similarly, in skeletal muscle, enhanced MAM formation may be beneficial in the early stage of diabetes, whereas continuous MAM enhancement aggravates insulin resistance. Furthermore, recent studies have suggested that ER stress may be the primary pathway through which MAMs induce brain insulin resistance, especially in the hypothalamus. This review will discuss the possible mechanisms underlying MAM-associated insulin resistance as well as the therapeutic potential of targeting the MAM in the treatment of type 2 diabetes.

Mitochondria-Associated Endoplasmic Reticulum Membranes (MAMs) and Their Prospective Roles in Kidney Disease.

Mitochondria-associated endoplasmic reticulum (ER) membranes (MAMs) serve as essential hubs for interorganelle communication in eukaryotic cells and play multifunctional roles in various biological pathways. A defect in ER-mitochondria signaling or MAMs dysfunction has pleiotropic effects on a variety of intracellular events, which results in disturbances of the mitochondrial quality control system, Ca2+ dyshomeostasis, apoptosis, ER stress, and inflammasome activation, which all contribute to the onset and progression of kidney disease. Here, we review the structure and molecular compositions of MAMs as well as the experimental methods used to study these interorganellar contact sites. We will specifically summarize the downstream signaling pathways regulated by MAMs, mainly focusing on mitochondrial quality control, oxidative stress, ER-mitochondria Ca2+ crosstalk, apoptosis, inflammasome activation, and ER stress. Finally, we will discuss how alterations in MAMs integrity contribute to the pathogenesis of kidney disease and offer directions for future research.

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.

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.

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

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. 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. 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. 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.

Feature Article: mTOR complex 2-Akt signaling at mitochondria-associated endoplasmic reticulum membranes (MAM) regulates mitochondrial physiology.

The target of rapamycin (TOR) is a highly conserved protein kinase and a central controller of growth. Mammalian TOR complex 2 (mTORC2) regulates AGC kinase family members and is implicated in various disorders, including cancer and diabetes. Here we report that mTORC2 is localized to the endoplasmic reticulum (ER) subcompartment termed mitochondria-associated ER membrane (MAM). mTORC2 localization to MAM was growth factor-stimulated, and mTORC2 at MAM interacted with the IP3 receptor (IP3R)-Grp75-voltage-dependent anion-selective channel 1 ER-mitochondrial tethering complex. mTORC2 deficiency disrupted MAM, causing mitochondrial defects including increases in mitochondrial membrane potential, ATP production, and calcium uptake. mTORC2 controlled MAM integrity and mitochondrial function via Akt mediated phosphorylation of the MAM associated proteins IP3R, Hexokinase 2, and phosphofurin acidic cluster sorting protein 2. Thus, mTORC2 is at the core of a MAM signaling hub that controls growth and metabolism.