Myocardial transcriptomic analysis of diabetic patients with aortic stenosis: key role for mitochondrial calcium signaling.

BACKGROUND: Type 2 diabetes (T2D) is a frequent comorbidity encountered in patients with severe aortic stenosis (AS), leading to an adverse left ventricular (LV) remodeling and dysfunction. Metabolic alterations have been suggested as contributors of the deleterious effect of T2D on LV remodeling and function in patients with severe AS, but so far, the underlying mechanisms remain unclear. Mitochondria play a central role in the regulation of cardiac energy metabolism. OBJECTIVES: We aimed to explore the mitochondrial alterations associated with the deleterious effect of T2D on LV remodeling and function in patients with AS, preserved ejection fraction, and no additional heart disease. METHODS: We combined an in-depth clinical, biological and echocardiography phenotype of patients with severe AS, with (n = 34) or without (n = 50) T2D, referred for a valve replacement, with transcriptomic and histological analyses of an intra-operative myocardial LV biopsy. RESULTS: T2D patients had similar AS severity but displayed worse cardiac remodeling, systolic and diastolic function than non-diabetics. RNAseq analysis identified 1029 significantly differentially expressed genes. Functional enrichment analysis revealed several T2D-specific upregulated pathways despite comorbidity adjustment, gathering regulation of inflammation, extracellular matrix organization, endothelial function/angiogenesis, and adaptation to cardiac hypertrophy. Downregulated gene sets independently associated with T2D were related to mitochondrial respiratory chain organization/function and mitochondrial organization. Generation of causal networks suggested a reduced Ca2+ signaling up to the mitochondria, with the measured gene remodeling of the mitochondrial Ca2+ uniporter in favor of enhanced uptake. Histological analyses supported a greater cardiomyocyte hypertrophy and a decreased proximity between the mitochondrial VDAC porin and the reticular IP3-receptor in T2D. CONCLUSIONS: Our data support a crucial role for mitochondrial Ca2+ signaling in T2D-induced cardiac dysfunction in severe AS patients, from a structural reticulum-mitochondria Ca2+ uncoupling to a mitochondrial gene remodeling. Thus, our findings open a new therapeutic avenue to be tested in animal models and further human cardiac biopsies in order to propose new treatments for T2D patients suffering from AS. TRIAL REGISTRATION: URL: https://www. CLINICALTRIALS: gov ; Unique Identifier: NCT01862237.

FLT3-ITD regulation of the endoplasmic reticulum functions in acute myeloid leukemia.

The FLT3-ITD mutation represents the most frequent genetic alteration in newly diagnosed acute myeloid leukemia (AML) patient and is associated with poor prognosis. Mutation result in the retention of a constitutively active form of this receptor in the endoplasmic reticulum (ER) and the subsequent modification of its downstream effectors. Here, we assessed the impact of such retention on ER homeostasis and found that mutant cells present lower levels of ER stress due to the overexpression of ERO1α, one of the main proteins of the protein folding machinery at the ER. Overexpression of ERO1α resulted essential for ITD mutant cells survival and chemoresistance and also played a crucial role in shaping the type of glucose metabolism in AML cells, being the mitochondrial pathway the predominant one in those with a higher ER stress (non-mutated cells) and the glycolytic pathway the predominant one in those with lower ER stress (mutated cells). Our data indicate that FLT3 mutational status dictates the route for glucose metabolism in an ERO1α depending on manner and this provides a survival advantage to tumors carrying these ITD mutations.

Endothelial Mitochondria-Associated Membranes (MAMs) Isolation by Percoll Step Gradients.

Mitochondria-associated membranes (MAMs) are regions where the endoplasmic reticulum (ER) interacts with mitochondria and regulate lipid trafficking, calcium signaling, ER stress, and inflammation activation. Isolation of MAMs from endothelial cells is vital for studying insight into the immune regulation of many inflammatory diseases. Endothelial cells (ECs) are critical innate immune cells due to their paracrine function of secreting interleukins, chemokines, cytokines, and growth factors, as well as expressing levels of pattern recognition receptors including toll-like receptors (TLRs). Furthermore, ECs regulate and recruit monocytes by expressing adhesion molecules including vascular cell adhesion molecule-1 (VCAM-1), intercellular adhesion molecule-1 (ICAM-1), P-selectin, and E-selectin, to facilitate monocyte diapedesis in areas of damage and inflammation. This protocol consists of step-by-step instructions on isolating pure MAMs and other subcellular fractions from endothelial cells, which is critical to understanding ER and mitochondria crosstalks in endothelial functions in health and disease.

Proteomic analysis of mitochondria associated membranes in renal ischemic reperfusion injury.

BACKGROUND: The mitochondria and endoplasmic reticulum (ER) communicate via contact sites known as mitochondria associated membranes (MAMs). Many important cellular functions such as bioenergetics, mitophagy, apoptosis, and calcium signaling are regulated by MAMs, which are thought to be closely related to ischemic reperfusion injury (IRI). However, there exists a gap in systematic proteomic research addressing the relationship between these cellular processes. METHODS: A 4D label free mass spectrometry-based proteomic analysis of mitochondria associated membranes (MAMs) from the human renal proximal tubular epithelial cell line (HK-2 cells) was conducted under both normal (N) and hypoxia/reperfusion (HR) conditions. Subsequent differential proteins analysis aimed to characterize disease-relevant signaling molecules. Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) analysis was applied to total proteins and differentially expressed proteins, encompassing Biological Process (BP), Cell Component (CC), Molecular Function (MF), and KEGG pathways. Further, Protein-Protein Interaction Network (PPI) exploration was carried out, leading to the identification of hub genes from differentially expressed proteins. Notably, Mitofusion 2 (MFN2) and BCL2/Adenovirus E1B 19-kDa interacting protein 3(BNIP3) were identified and subsequently validated both in vitro and in vivo. Finally, the impact of MFN2 on MAMs during hypoxia/reoxygenation was explored through regulation of gene expression. Subsequently, a comparative proteomics analysis was conducted between OE-MFN2 and normal HK-2 cells, providing further insights into the underlying mechanisms. RESULTS: A total of 4489 proteins were identified, with 3531 successfully quantified. GO/KEGG analysis revealed that MAM proteins were primarily associated with mitochondrial function and energy metabolism. Differential analysis between the two groups showed that 688 proteins in HR HK-2 cells exhibited significant changes in expression level with P-value < 0.05 and HR/N > 1.5 or HR/N < 0.66 set as the threshold criteria. Enrichment analysis of differentially expressed proteins unveiled biological processes such as mRNA splicing, apoptosis regulation, and cell division, while molecular functions were predominantly associated with energy metabolic activity. These proteins play key roles in the cellular responses during HR, offering insights into the IRI mechanisms and potential therapeutic targets. The validation of hub genes MFN2 and BNIP3 both in vitro and vivo was consistent with the proteomic findings. MFN2 demonstrated a protective role in maintaining the integrity of mitochondria associated membranes (MAMs) and mitigating mitochondrial damage following hypoxia/reoxygenation injury, this protective effect may be associated with the activation of the PI3K/AKT pathway. CONCLUSIONS: The proteins located in mitochondria associated membranes (MAMs) are implicated in crucial roles during renal ischemic reperfusion injury (IRI), with MFN2 playing a pivotal regulatory role in this context.

Mitochondria-Associated Membranes as Key Regulators in Cellular Homeostasis and the Potential Impact of Exercise on Insulin Resistance.

The communication between mitochondria and the endoplasmic reticulum (ER) is facilitated by a dynamic membrane structure formed by protein complexes known as mitochondria-associated membranes (MAMs). The structural and functional integrity of MAMs is crucial for insulin signal transduction, relying heavily on their regulation of intracellular calcium homeostasis, lipid homeostasis, mitochondrial quality control, and endoplasmic reticulum stress (ERS). This article reviews recent research findings, suggesting that exercise may promote the remodeling of MAMs structure and function by modulating the expression of molecules associated with their structure and function. This, in turn, restores cellular homeostasis and ultimately contributes to the amelioration of insulin resistance (IR). These insights provide additional possibilities for the study and treatment of insulin resistance-related metabolic disorders such as obesity, diabetes, fatty liver, and atherosclerosis.

Mlg1, a yeast acyltransferase located in ER membranes associated with mitochondria (MAMs), is involved in de novo synthesis and remodelling of phospholipids.

In cells, phospholipids contain acyl chains of variable lengths and saturation, features that affect their functions. Their de novo synthesis in the endoplasmic reticulum takes place via the cytidine diphosphate diacylglycerol (CDP-DAG) and Kennedy pathways, which are conserved in eukaryotes. PA is a key intermediate for all phospholipids (PI, PIPs, PS, PE, PC, PG and CL). The de novo synthesis of PA occurs by acylation of glycerophosphate leading to the synthesis of 1-acyl lysoPA and subsequent acylation of 1-acyl lysoPA at the sn-2 position. Using membranes from Escherichia coli overexpressing MLG1, we showed that the yeast gene MLG1 encodes an acyltransferase, leading specifically to the synthesis of PA from 1-acyl lysoPA. Moreover, after their de novo synthesis, phospholipids can be remodelled by acyl exchange with one and/or two acyl chains exchanged at the sn-1 and/or sn-2 position. Based on shotgun lipidomics of the reference and mlg1Δ strains, as well as biochemical assays for acyltransferase activities, we identified an additional remodelling activity for Mlg1p, namely, incorporation of palmitic acid into the sn-1 position of PS and PE. By using confocal microscopy and subcellular fractionation, we also found that this acyltransferase is located in ER membranes associated with mitochondria, a finding that highlights the importance of these organelles in the global cellular metabolism of lipids.

Identification of myoferlin as a mitochondria-associated membranes component required for calcium signaling in PDAC cell lines.

BACKGROUND: Pancreatic ductal adenocarcinoma is an aggressive cancer type with one of the lowest survival rates due to late diagnosis and the absence of effective treatments. A better understanding of PDAC biology will help researchers to discover the Achilles' heel of cancer cells. In that regard, our research team investigated the function of an emerging oncoprotein known as myoferlin. Myoferlin is overexpressed in PDAC and its silencing/targeting has been shown to affect cancer cell proliferation, migration, mitochondrial dynamics and metabolism. Nevertheless, our comprehension of myoferlin functions in cells remains limited. In this study, we aimed to understand the molecular mechanism linking myoferlin silencing to mitochondrial dynamics. METHODS: Experiments were performed on two pancreas cancer cell lines, Panc-1 and MiaPaCa-2. Myoferlin localization on mitochondria was evaluated by immunofluorescence, proximity ligation assay, and cell fractionation. The presence of myoferlin in mitochondria-associated membranes was assessed by cell fractionation and its function in mitochondrial calcium transfer was evaluated using calcium flow experiments, proximity ligation assays, co-immunoprecipitation, and timelapse fluorescence microscopy in living cells. RESULTS: Myoferlin localization on mitochondria was investigated. Our results suggest that myoferlin is unlikely to be located on mitochondria. Instead, we identified myoferlin as a new component of mitochondria-associated membranes. Its silencing significantly reduces the mitochondrial calcium level upon stimulation, probably through myoferlin interaction with the inositol 1,4,5-triphosphate receptors 3. CONCLUSIONS: For the first time, myoferlin was specifically demonstrated to be located in mitochondria-associated membranes where it participates to calcium flow. We hypothesized that this function explains our previous results on mitochondrial dynamics. This study improves our comprehension of myoferlin localization and function in cancer biology.

RTN3 deficiency exacerbates cisplatin-induced acute kidney injury through the disruption of mitochondrial stability.

Reticulum 3 (RTN3) is an endoplasmic reticulum (ER) protein that has been reported to act in neurodegenerative diseases and lipid metabolism. However, the role of RTN3 in acute kidney injury (AKI) has not been explored. Here, we employed public datasets, patient data, and animal models to explore the role of RTN3 in AKI. The underlying mechanisms were studied in primary renal tubular epithelial cells and in the HK2 cell line. We found reduced expression of RTN3 in AKI patients, cisplatin-induced mice, and cisplatin-treated HK2 cells. RTN3-null mice exhibit more severe AKI symptoms and kidney fibrosis after cisplatin treatment. Mitochondrial dysfunction was also found in cells with RTN3 knockdown or knockout. A mechanistic study revealed that RTN3 can interact with HSPA9 in kidney cells. RTN3 deficiency may disrupt the RTN3-HSPA9-VDAC2 complex and affect MAMs during ER-mitochondrion contact, which further leads to mitochondrial dysfunction and exacerbates cisplatin-induced AKI. Our study indicated that RTN3 was important in the kidney and that a decrease in RTN3 in the kidney might be a risk factor for the aggravation of AKI.

The SIRT3-ATAD3A axis regulates MAM dynamics and mitochondrial calcium homeostasis in cardiac hypertrophy.

Mitochondria are energy-producing organelles that are mobile and harbor dynamic network structures. Although mitochondria and endoplasmic reticulum (ER) play distinct cellular roles, they are physically connected to maintain functional homeostasis. Abnormal changes in this interaction have been linked to pathological states, including cardiac hypertrophy. However, the exact regulatory molecules and mechanisms are yet to be elucidated. Here, we report that ATPase family AAA-domain containing protein 3A (ATAD3A) is an essential regulator of ER-mitochondria interplay within the mitochondria-associated membrane (MAM). ATAD3A prevents isoproterenol (ISO)-induced mitochondrial calcium accumulation, improving mitochondrial dysfunction and ER stress, which preserves cardiac function and attenuates cardiac hypertrophy. We also find that ATAD3A is a new substrate of NAD+-dependent deacetylase Sirtuin 3 (SIRT3). Notably, the heart mitochondria of SIRT3 knockout mice exhibited excessive formation of MAMs. Mechanistically, ATAD3A specifically undergoes acetylation, which reduces self-oligomerization and promotes cardiac hypertrophy. ATAD3A oligomerization is disrupted by acetylation at K134 site, and ATAD3A monomer closely interacts with the IP3R1-GRP75-VDAC1 complex, which leads to mitochondrial calcium overload and dysfunction. In summary, ATAD3A localizes to the MAMs, where it protects the homeostasis of ER-mitochondria contacts, quenching mitochondrial calcium overload and keeping mitochondrial bioenergetics unresponsive to ER stress. The SIRT3-ATAD3A axis represents a potential therapeutic target for cardiac hypertrophy.

Mitochondrial influences on smooth muscle phenotype.

Smooth muscle cells transition reversibly between contractile and noncontractile phenotypes in response to diverse influences, including many from mitochondria. Numerous molecules including myocardin, procontractile miRNAs, and the mitochondrial protein prohibitin-2 promote contractile differentiation; this is opposed by mitochondrial reactive oxygen species (mtROS), high lactate concentrations, and metabolic reprogramming induced by mitophagy and/or mitochondrial fission. A major pathway through which vascular pathologies such as oncogenic transformation, pulmonary hypertension, and atherosclerosis cause loss of vascular contractility is by enhancing mitophagy and mitochondrial fission with secondary effects on smooth muscle phenotype. Proproliferative miRNAs and the mitochondrial translocase TOMM40 also attenuate contractile differentiation. Hypoxia can initiate loss of contractility by enhancing mtROS and lactate production while simultaneously depressing mitochondrial respiration. Mitochondria can reduce cytosolic calcium by moving it across the inner mitochondrial membrane via the mitochondrial calcium uniporter, and then through mitochondria-associated membranes to and from calcium stores in the sarcoplasmic/endoplasmic reticulum. Through these effects on calcium, mitochondria can influence multiple calcium-sensitive nuclear transcription factors and genes, some of which govern smooth muscle phenotype, and possibly also the production of genomically encoded mitochondrial proteins and miRNAs (mitoMirs) that target the mitochondria. In turn, mitochondria also can influence nuclear transcription and mRNA processing through mitochondrial retrograde signaling, which is currently a topic of intensive investigation. Mitochondria also can signal to adjacent cells by contributing to the content of exosomes. Considering these and other mechanisms, it is becoming increasingly clear that mitochondria contribute significantly to the regulation of smooth muscle phenotype and differentiation.

Methane-Rich Saline Suppresses ER-Mitochondria Contact and Activation of the NLRP3 Inflammasome by Regulating the PERK Signaling Pathway to Ameliorate Intestinal Ischemia‒Reperfusion Injury.

Intestinal ischemia‒reperfusion (I/R) injury is a common pathological process in patients undergoing gastrointestinal surgery, leading to local intestinal damage and increased microvascular permeability, eventually causing extraintestinal multiple organ dysfunction or sepsis. The NLRP3-mediated inflammatory response is associated with I/R injury. Methane saline (MS) has anti-pyroptosis properties. This study aims to explore the protective effect of MS on intestinal I/R injury and its potential mechanisms. After MS pretreatment, the in vivo model was established by temporarily clipping the mouse superior mesentery artery with a noninvasive vascular clamp, and the in vitro model was established by OGD/R on Caco-2 cells. The results of HE and TUNEL staining showed intestinal barrier damage after I/R injury, which was consistent with the IHC staining results of tight junction proteins. Moreover, the expression of the NLRP3 signaling pathway was increased after I/R injury, and inhibition of NLRP3 activation reduced Caco-2 cell injury, indicating that NLRP3-mediated pyroptosis was one of the main forms of cell death after I/R injury. Subsequently, we found that MS treatment ameliorated intestinal barrier function after I/R injury by suppressing NLRP3-mediated pyroptosis. MS treatment also reduced mitochondria-associated membrane (MAM) formation, which was considered to be a platform for activation of the NLRP3 inflammasome. Importantly, MS reduced ER stress, which was related to the PERK signaling pathway. Knocking down PERK, a key protein involved in ER stress and MAM formation, reversed the protective effect of MS, indicating that MS suppressed NLRP3 by reducing ER stress and MAM formation. In conclusion, we believe that MS suppresses MAMs and activation of the NLRP3 inflammasome by regulating the PERK signaling pathway to ameliorate intestinal I/R injury.

Ultrastructural Abnormalities in Induced Pluripotent Stem Cell-Derived Neural Stem Cells and Neurons of Two Cohen Syndrome Patients.

Cohen syndrome is an autosomal recessive disorder caused by VPS13B (COH1) gene mutations. This syndrome is significantly underdiagnosed and is characterized by intellectual disability, microcephaly, autistic symptoms, hypotension, myopia, retinal dystrophy, neutropenia, and obesity. VPS13B regulates intracellular membrane transport and supports the Golgi apparatus structure, which is critical for neuron formation. We generated induced pluripotent stem cells from two patients with pronounced manifestations of Cohen syndrome and differentiated them into neural stem cells and neurons. Using transmission electron microscopy, we documented multiple new ultrastructural changes associated with Cohen syndrome in the neuronal cells. We discovered considerable disturbances in the structure of some organelles: Golgi apparatus fragmentation and swelling, endoplasmic reticulum structural reorganization, mitochondrial defects, and the accumulation of large autophagosomes with undigested contents. These abnormalities underline the ultrastructural similarity of Cohen syndrome to many neurodegenerative diseases. The cell models that we developed based on patient-specific induced pluripotent stem cells can serve to uncover not only neurodegenerative processes, but the causes of intellectual disability in general.

Screening of Crucial Cytosolicproteins Interconnecting the Endoplasmic Reticulum and Mitochondria in Parkinson's Disease and the Impact of Anti-Parkinson Drugs in the Preservation of Organelle Connectivity.

Mitochondrial dysfunction is well-established in Parkinson's disease (PD); however, its dysfunctions associating with cell organelle connectivity remain unknown. We aimed to establish the crucial cytosolic protein involved in organelle connectivity between mitochondria and the endopalmic reticulum (ER) through a computational approach by constructing an organelle protein network to extract functional clusters presenting the crucial PD protein connecting organelles. Then, we assessed the influence of anti-parkinsonism drugs (n = 35) on the crucial protein through molecular docking and molecular dynamic simulation and further validated its gene expression in PD participants under, istradefylline (n = 25) and amantadine (n = 25) treatment. Based on our investigation, D-aspartate oxidase (DDO )protein was found to be the critical that connects both mitochondria and the ER. Further, molecular docking showed that istradefylline has a high affinity (-9.073 kcal/mol) against DDO protein, which may disrupt mitochondrial-ER connectivity. While amantadine (-4.53 kcal/mol) shows negligible effects against DDO that contribute to conformational changes in drug binding, Successively, DDO gene expression was downregulated in istradefylline-treated PD participants, which elucidated the likelihood of an istradefylline off-target mechanism. Overall, our findings illuminate the off-target effects of anti-parkinsonism medications on DDO protein, enabling the recommendation of off-target-free PD treatments.

Hepatic levels of S-adenosylmethionine regulate the adaptive response to fasting.

There has been an intense focus to uncover the molecular mechanisms by which fasting triggers the adaptive cellular responses in the major organs of the body. Here, we show that in mice, hepatic S-adenosylmethionine (SAMe)-the principal methyl donor-acts as a metabolic sensor of nutrition to fine-tune the catabolic-fasting response by modulating phosphatidylethanolamine N-methyltransferase (PEMT) activity, endoplasmic reticulum-mitochondria contacts, ß-oxidation, and ATP production in the liver, together with FGF21-mediated lipolysis and thermogenesis in adipose tissues. Notably, we show that glucagon induces the expression of the hepatic SAMe-synthesizing enzyme methionine adenosyltransferase α1 (MAT1A), which translocates to mitochondria-associated membranes. This leads to the production of this metabolite at these sites, which acts as a brake to prevent excessive ß-oxidation and mitochondrial ATP synthesis and thereby endoplasmic reticulum stress and liver injury. This work provides important insights into the previously undescribed function of SAMe as a new arm of the metabolic adaptation to fasting.

Presenilins and mitochondria-an intriguing link: mini-review.

This review uncovers the intricate relationship between presenilins, calcium, and mitochondria in the context of Alzheimer's disease (AD), with a particular focus on the involvement of presenilin mutations in mitochondrial dysfunction. So far, it is unclear whether the impairment of mitochondrial function arises primarily from damage inflicted by ß-amyloid upon mitochondria or from the disruption of calcium homeostasis due to presenilins dysfunctions. The roles of presenilins in mitophagy, autophagy, mitochondrial dynamics, and many other functions, non-γ-secretase related, also require close attention in future research. Resolution of contradictions in understanding of presenilins cellular functions are needed for new effective therapeutic strategies for AD.

The ER-mitochondria interface, where Ca2+ and cell death meet.

Endoplasmic reticulum (ER)-mitochondria contact sites are crucial to allow Ca2+ flux between them and a plethora of proteins participate in tethering both organelles together. Inositol 1,4,5-trisphosphate receptors (IP3Rs) play a pivotal role at such contact sites, participating in both ER-mitochondria tethering and as Ca2+-transport system that delivers Ca2+ from the ER towards mitochondria. At the ER-mitochondria contact sites, the IP3Rs function as a multi-protein complex linked to the voltage-dependent anion channel 1 (VDAC1) in the outer mitochondrial membrane, via the chaperone glucose-regulated protein 75 (GRP75). This IP3R-GRP75-VDAC1 complex supports the efficient transfer of Ca2+ from the ER into the mitochondrial intermembrane space, from which the Ca2+ ions can reach the mitochondrial matrix through the mitochondrial calcium uniporter. Under physiological conditions, basal Ca2+ oscillations deliver Ca2+ to the mitochondrial matrix, thereby stimulating mitochondrial oxidative metabolism. However, when mitochondrial Ca2+ overload occurs, the increase in [Ca2+] will induce the opening of the mitochondrial permeability transition pore, thereby provoking cell death. The IP3R-GRP75-VDAC1 complex forms a hub for several other proteins that stabilize the complex and/or regulate the complex's ability to channel Ca2+ into the mitochondria. These proteins and their mechanisms of action are discussed in the present review with special attention for their role in pathological conditions and potential implication for therapeutic strategies.

AMPK-endoplasmic reticulum stress axis contributes to lipopolysaccharide-caused mitochondrial dysfunction by regulating mitochondria-associated membrane function in bovine hepatocytes.

Mitochondrial homeostasis is closely associated with cellular homeostasis process, whereas mitochondrial dysfunction contributes to apoptosis and mitophagy. Hence, analyzing the mechanism of lipopolysaccharide (LPS)-caused mitochondrial damage is necessary to understand how cellular homeostasis is maintained in bovine hepatocytes. Mitochondria-associated membranes (MAM), a connection between endoplasmic reticulum (ER) and mitochondria, is important to control mitochondrial function. To investigate the underlying mechanisms of the LPS-caused mitochondrial dysfunction, hepatocytes isolated from dairy cows at ∼160 d in milk (DIM) were pretreated with the specific inhibitors of adenosine 5'-monophosphate-activated protein kinase (AMPK), ER stress, RNA-activated protein kinase-like ER kinase (PERK), inositol-requiring enzyme 1α (IRE1α), c-Jun N-terminal kinase, and autophagy followed by a 12 I1/4g/mL LPS treatment. The results showed that inhibiting ER stress with 4-phenylbutyric acid decreased the levels of autophagy and mitochondrial damage with AMPK inactivation in LPS-treated hepatocytes. The AMPK inhibitor compound C pretreatment alleviated LPS-induced ER stress, autophagy and mitochondrial dysfunction by regulating the expression of MAM-related genes, such as mitofusin 2 (MFN2), PERK, and IRE1α. Moreover, inhibiting PERK and IRE1α mitigated autophagy and mitochondrial dynamic disruption by regulating the MAM function. Additionally, blocking c-Jun N-terminal kinase, the downstream sensor of IRE1α, could reduce the levels of autophagy and apoptosis and restore the balance of mitochondrial fusion and fission by modulating the B cell leukemia 2 (BCL-2)/BCL-2 interacting protein 1 (BECLIN1) complex in the LPS-treated bovine hepatocytes. Furthermore, autophagy blockage with chloroquine could intervene in LPS-caused apoptosis to restore mitochondrial function. Collectively, these findings suggest that the AMPK-ER stress axis is involved in the LPS-caused mitochondrial dysfunction by mediating the MAM activity in bovine hepatocytes.

Phospholipid metabolism in the liver - Implications for phosphatidylserine in non-alcoholic fatty liver disease.

Mammalian cells contain more than a thousand different glycerophospholipid species that are essential membrane components and signalling molecules, with phosphatidylserine (PS) giving membranes their negative surface charge. Depending on the tissue, PS is important in apoptosis, blood clotting, cancer pathogenesis, as well as muscle and brain function, processes that are dependent on the asymmetrical distribution of PS on the plasma membrane and/or the capacity of PS to act as anchorage for various signalling proteins. Recent studies have implicated hepatic PS in the progression of non-alcoholic fatty liver disease (NAFLD), either as beneficial in the context of suppressing hepatic steatosis and fibrosis, or on the other hand as a potential contributor to the progression of liver cancer. This review provides an extensive overview of hepatic phospholipid metabolism, including its biosynthetic pathways, intracellular trafficking and roles in health and disease, further taking a deeper dive into PS metabolism, including associate and causative evidence of the role of PS in advanced liver disease.

Mitochondria-SR interaction and mitochondrial fusion/fission in the regulation of skeletal muscle metabolism.

Mitochondria-endoplasmic/sarcoplasmic reticulum (ER/SR) interaction and mitochondrial fusion/fission are critical processes that influence substrate oxidation. This narrative review summarizes the evidence on the effects of substrate availability on mitochondrial-SR interaction and mitochondria fusion/fission dynamics to modulate substrate oxidation in human skeletal muscle. Evidence shows that an increase in mitochondria-SR interaction and mitochondrial fusion are associated with elevated fatty acid oxidation. In contrast, a decrease in mitochondria-SR interaction and an increase in mitochondrial fission are associated with an elevated glycolytic activity. Based on the evidence reviewed, we postulate two hypotheses for the link between mitochondrial dynamics and insulin resistance in human skeletal muscle. First, glucose and fatty acid availability modifies mitochondria-SR interaction and mitochondrial fusion/fission to help the cell to adapt substrate oxidation appropriately. Individuals with an impaired response to these substrate challenges will accumulate lipid species and develop insulin resistance in skeletal muscle. Second, a chronically elevated substrate availability (e.g. overfeeding) increases mitochondrial production of reactive oxygen species and induced mitochondrial fission. This decreases fatty acid oxidation, thus leading to the accumulation of lipid species and insulin resistance in skeletal muscle. Altogether, we propose mitochondrial dynamics as a potential target for disturbances associated with low fatty acid oxidation.

The spatial expression of mTORC2-AKT-IP3R signal pathway in mitochondrial combination of endoplasmic reticulum of maternal fetal interface trophoblast in intrahepatic cholestasis of pregnancy.

OBJECTIVES: Intrahepatic cholestasis of pregnancy (ICP) is complicated by adverse fetal outcomes and even fetal death, the mechanism remains unclear. This study aims at evaluating the differential expression of mTORC2-AKT-IP3R signaling pathway, which accurately regulate Ca2+ transfer across mitochondria-associated membranes (MAMs) and determine the stress intensity experienced by endoplasmic reticulum (ER) and mitochondria, in patients diagnosed with ICP. METHODS: We combined western blot analysis and placental immunofluorescence co-localization detection to assess the expression and co-localization of the mTORC2-AKT-IP3R signaling pathway in severe (maternal total bile acid (TBA) levels ≥40 µmol/L) and mild (maternal TBA 10-40 µmol/L) ICP. RESULTS: Compared with the control and mild ICP groups, phosphorylated protein kinase B (p-AKT) levels were significantly upregulated in the severe ICP group. Placental Rictor levels were lower in the mild ICP group than in the control group and were further downregulated in the severe ICP group. IP3R3 and p-IP3R3 levels were lower in placentas in the severe ICP group than in those in the mild ICP and control groups. Moreover, the co-localization of IP3R3 and p-AKT in patients in the mild and severe ICP groups was significantly elevated compared with that in patients in the control group. CONCLUSIONS: In patients with severe ICP, limited expression of Rictor and elevated p-AKT levels would suppress IP3R3/p-IP3R3 levels in MAMs. This inhibition might influence the transportation of Ca2+ from the ER to the mitochondria, thus weaken the stress adaptation associated with MAMs. Our results reveal the possible pathophysiological mechanism of adverse fetal outcomes in ICP.