Non-alcoholic fatty liver disease in mice with hepatocyte-specific deletion of mitochondrial fission factor

Abstract Aims/hypothesis Mitochondria are highly dynamic organelles continuously undergoing fission and fusion, referred to as mitochondrial dynamics, to adapt to nutritional demands. Evidence suggests that impaired mitochondrial dynamics leads to metabolic abnormalities such as non-alcoholic steatohepatitis (NASH) phenotypes. However, how mitochondrial dynamics are involved in the development of NASH is poorly understood. This study aimed to elucidate the role of mitochondrial fission factor (MFF) in the development of NASH. Methods We created mice with hepatocyte-specific deletion of MFF (MffLiKO). MffLiKO mice fed normal chow diet (NCD) or high-fat diet (HFD) were evaluated for metabolic variables and their livers were examined by histological analysis. To elucidate the mechanism of development of NASH, we examined the expression of genes related to endoplasmic reticulum (ER) stress and lipid metabolism, and the secretion of triacylglycerol (TG) using the liver and primary hepatocytes isolated from MffLiKO and control mice. Results MffLiKO mice showed aberrant mitochondrial morphologies with no obvious NASH phenotypes during NCD, while they developed full-blown NASH phenotypes in response to HFD. Expression of genes related to ER stress was markedly upregulated in the liver from MffLiKO mice. In addition, expression of genes related to hepatic TG secretion was downregulated, with reduced hepatic TG secretion in MffLiKO mice in vivo and in primary cultures of MFF-deficient hepatocytes in vitro. Furthermore, thapsigargin-induced ER stress suppressed TG secretion in primary hepatocytes isolated from control mice. Conclusions/interpretation We demonstrated that ablation of MFF in liver provoked ER stress and reduced hepatic TG secretion in vivo and in vitro. Moreover, MffLiKO mice were more susceptible to HFD-induced NASH phenotype than control mice, partly because of ER stress-induced apoptosis of hepatocytes and suppression of TG secretion from hepatocytes. This study provides evidence for the role of mitochondrial fission in the development of NASH. Graphical abstract

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Abstract 269: Cardiac Specific Disruption of Drp-1 Causes the Development of Cardiac Dysfunction via Inhibition of Autophagy and Induction of Apoptosis

Circulation Research ◽

10.1161/res.113.suppl_1.a269 ◽

2013 ◽

Vol 113 (suppl_1) ◽

Author(s):

Yoshiyuki Ikeda ◽

Junichi Sadoshima

Keyword(s):

Mitochondrial Dysfunction ◽

Systolic Function ◽

Mitochondrial Fission ◽

Mitochondrial Mass ◽

Reduced Ejection Fraction ◽

Tibia Length ◽

Cardiac Systolic Function

Fission and fusion affect mitochondrial turnover in part by modulating mitophagy. This study aimed to clarify the role of mitochondrial fission in regulating cardiac function and autophagy in the heart. Dynamin-related protein 1 (Drp-1) plays an essential role in mediating mitochondrial fission. Therefore, we generated cardiac specific Drp-1 KO mice and utilized cultured cardiomyocytes transduced with adenovirus harboring short hairpin Drp-1 (Ad-shDrp-1) to test the effect of Drp-1 disruption both in vivo and in vitro. In Drp-1 KO hearts we observed a significantly greater mitochondrial mass ratio compared to control, as assessed by electron microscopy (Drp-1 KO: 3.57 ± 1.38, control: 1.18 ± 0.31, P<0.05). Mitochondrial ATP content was significantly lower (0.70 ± 0.07 vs 1.03 ± 0.10, P<0.05), while mitochondrial swelling was significantly greater (% decrease in absorbance; 8.01 ± 1.99 vs 2.01 ± 0.58, P<0.05) in Drp-1 KO hearts versus control. Mitochondrial membrane potential, assessed by JC-1 staining, was significantly reduced in myocytes with knockdown of Drp-1. Taken together, these results suggest that inhibition of fission causes mitochondrial dysfunction. We also examined the effect of Drp-1 depletion on autophagy. We found that the amount of LC-3 II was significantly less (0.47 ± 0.16 vs 1.32 ±0.34, P<0.05), whereas p62 expression was significantly greater (1.14 ± 0.16 vs 0.16 ± 0.06, P<0.01) in Drp-1 KO hearts compared to control. The number of LC3 dots in Ad-shDrp-1 transduced myocytes was lower than that of sh-scramble treatment. We investigated apoptosis and found that the amount of cleaved caspase-3 (0.62 ± 0.24 vs 0.18 ± 0.04, P<0.05) and the number of TUNEL positive cells (0.22 ± 0.12 vs 0.03 ± 0.06%, P<0.01) were higher in Drp-1 KO versus control hearts. Cardiac systolic function was reduced (ejection fraction; 44.5 ± 6.3 vs 85.4 ± 5.7%, P<0.01) and LVW/tibia length was greater (4.48 ± 0.38 vs 3.84 ± 0.58, P<0.05) in Drp-1 KO mice compared to control. Finally, we observed that the survival rate of Drp-1 KO mice was significantly reduced compared to control mice. Our results demonstrate that inhibition of mitochondrial fission via disruption of Drp-1 inhibits autophagy and causes mitochondrial dysfunction, thereby promoting cardiomyopathy.

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LncRNA NEAT1/microRNA-129-5p/SOCS2 axis regulates liver fibrosis in alcoholic steatohepatitis

Journal of Translational Medicine ◽

10.1186/s12967-020-02577-5 ◽

2020 ◽

Vol 18 (1) ◽

Author(s):

Junfeng Ye ◽

Yuanqiang Lin ◽

Ying Yu ◽

Di Sun

Keyword(s):

Liver Fibrosis ◽

Liver Function ◽

Inflammatory Factors ◽

Cytokine Signaling ◽

Alcoholic Fatty Liver ◽

Alcoholic Steatohepatitis ◽

Inflammation Reaction

Abstract Background Long non-coding RNA nuclear paraspeckle assembly transcript 1 (NEAT1) has been reported to play an essential role in non-alcoholic fatty liver disease. However, the role of NEAT1 in regulation of alcoholic steatohepatitis (ASH) remains largely unknown. This study aims to explore the role of NEAT1 in ASH by mediating microRNA-129-5p (miR-129-5p) targeting suppressor of cytokine signaling 2 (SOCS2). Methods NEAT1, miR-129-5p and SOCS2 expression in serum of ASH patients were assessed. In the in vitro cellular experiment, we transfected siRNAs, oligonucleotides or plasmids into ethanol-induced AML-12 mouse hepatocytes to alter NEAT1 and miR-129-5p expression, and inflammatory factors and lipid content were determined. In the in vivo animal experiment, we injected lentiviruses carrying siRNAs, oligonucleotides or plasmids onto ASH mice (ASH induced by feeding mice a Lieber-DeCarli ethanol diet) to alter NEAT1 and miR-129-5p expression through the tail vein. Serum liver function, blood lipids and inflammatory factors were detected; liver histopathology, liver cell apoptosis, and fibrosis were observed. The relationship between NEAT1 and miR-129-5p, or between miR-129-5p and SOCS2 was verified. Results MiR-129-5p was reduced while NEAT1 and SOCS2 were elevated in ASH. Inhibited NEAT1 or elevated miR-129-5p suppressed the elevated lipid metabolism and restrained inflammation reaction in ethanol-stimulated AML-12 cells. The promoted miR-129-5p and inhibited NEAT1 could improve the liver function and repress blood lipid, inflammation reaction, hepatocyte apoptosis and liver fibrosis in ethanol-induced ASH mice. Furthermore, NEAT1 could negatively regulate miR-129-5p to target SOCS2. Conclusion We have found that the inhibited NEAT1 could suppress liver fibrosis in ASH mice by promoting miR-129-5p and restraining SOCS2, thereby decelerating the development of ASH.

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Trophectoderm-Specific Knockdown of LIN28 Decreases Expression of Genes Necessary for Cell Proliferation and Reduces Elongation of Sheep Conceptus

International Journal of Molecular Sciences ◽

10.3390/ijms21072549 ◽

2020 ◽

Vol 21 (7) ◽

pp. 2549 ◽

Cited By ~ 1

Author(s):

Asghar Ali ◽

Mark Stenglein ◽

Thomas Spencer ◽

Gerrit Bouma ◽

Russell Anthony ◽

...

Keyword(s):

Cell Proliferation ◽

Critical Period ◽

In Vitro Experiments ◽

Placental Development ◽

Expression Of Genes ◽

Trophectoderm Cells ◽

Successful Establishment

LIN28 inhibits let-7 miRNA maturation which prevents cell differentiation and promotes proliferation. We hypothesized that the LIN28-let-7 axis regulates proliferation-associated genes in sheep trophectoderm in vivo. Day 9-hatched sheep blastocysts were incubated with lentiviral particles to deliver shRNA targeting LIN28 specifically to trophectoderm cells. At day 16, conceptus elongation was significantly reduced in LIN28A and LIN28B knockdowns. Let-7 miRNAs were significantly increased and IGF2BP1-3, HMGA1, ARID3B, and c-MYC were decreased in trophectoderm from knockdown conceptuses. Ovine trophoblast (OTR) cells derived from day 16 trophectoderm are a useful tool for in vitro experiments. Surprisingly, LIN28 was significantly reduced and let-7 miRNAs increased after only a few passages of OTR cells, suggesting these passaged cells represent a more differentiated phenotype. To create an OTR cell line more similar to day 16 trophectoderm we overexpressed LIN28A and LIN28B, which significantly decreased let-7 miRNAs and increased IGF2BP1-3, HMGA1, ARID3B, and c-MYC compared to control. This is the first study showing the role of the LIN28-let-7 axis in trophoblast proliferation and conceptus elongation in vivo. These results suggest that reduced LIN28 during early placental development can lead to reduced trophoblast proliferation and sheep conceptus elongation at a critical period for successful establishment of pregnancy.

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IκB kinase 2 is not essential for platelet activation

Blood Advances ◽

10.1182/bloodadvances.2019001044 ◽

2020 ◽

Vol 4 (4) ◽

pp. 638-643

Author(s):

Manuel Salzmann ◽

Sonja Bleichert ◽

Bernhard Moser ◽

Marion Mussbacher ◽

Mildred Haase ◽

...

Keyword(s):

Platelet Activation ◽

Active Site ◽

Bleeding Time ◽

Thrombus Formation ◽

Human Platelets ◽

Iκb Kinase ◽

Specific Deletion

Abstract Platelets are small anucleate cells that release a plethora of molecules to ensure functional hemostasis. It has been reported that IκB kinase 2 (IKK2), the central enzyme of the inflammatory NF-κB pathway, is involved in platelet activation, because megakaryocyte/platelet-specific deletion of exons 6 and 7 of IKK2 resulted in platelet degranulation defects and prolonged bleeding. We aimed to investigate the role of IKK2 in platelet physiology in more detail, using a platelet-specific IKK2 knockout via excision of exon 3, which makes up the active site of the enzyme. We verified the deletion on genomic and transcriptional levels in megakaryocytes and were not able to detect any residual IKK2 protein; however, platelets from these mice did not show any functional impairment in vivo or in vitro. Bleeding time and thrombus formation were not affected in platelet-specific IKK2-knockout mice. Moreover, platelet aggregation, glycoprotein GPIIb/IIIa activation, and degranulation were unaltered. These observations were confirmed by pharmacological inhibition of IKK2 with TPCA-1 and BMS-345541, which did not affect activation of murine or human platelets over a wide concentration range. Altogether, our results imply that IKK2 is not essential for platelet function.

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Talin is required for integrin-mediated platelet function in hemostasis and thrombosis

Journal of Experimental Medicine ◽

10.1084/jem.20071800 ◽

2007 ◽

Vol 204 (13) ◽

pp. 3103-3111 ◽

Cited By ~ 188

Author(s):

Brian G. Petrich ◽

Patrizia Marchese ◽

Zaverio M. Ruggeri ◽

Saskia Spiess ◽

Rachel A.M. Weichert ◽

...

Keyword(s):

Platelet Adhesion ◽

Ex Vivo ◽

Focal Adhesions ◽

Β1 Integrin ◽

Normal Morphology ◽

And Function ◽

Specific Deletion

Integrins are critical for hemostasis and thrombosis because they mediate both platelet adhesion and aggregation. Talin is an integrin-binding cytoplasmic adaptor that is a central organizer of focal adhesions, and loss of talin phenocopies integrin deletion in Drosophila. Here, we have examined the role of talin in mammalian integrin function in vivo by selectively disrupting the talin1 gene in mouse platelet precursor megakaryocytes. Talin null megakaryocytes produced circulating platelets that exhibited normal morphology yet manifested profoundly impaired hemostatic function. Specifically, platelet-specific deletion of talin1 led to spontaneous hemorrhage and pathological bleeding. Ex vivo and in vitro studies revealed that loss of talin1 resulted in dramatically impaired integrin αIIbβ3-mediated platelet aggregation and β1 integrin–mediated platelet adhesion. Furthermore, loss of talin1 strongly inhibited the activation of platelet β1 and β3 integrins in response to platelet agonists. These data establish that platelet talin plays a crucial role in hemostasis and provide the first proof that talin is required for the activation and function of mammalian α2β1 and αIIbβ3 integrins in vivo.

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PIM Kinases Alter Mitochondrial Dynamics and Chemosensitivity in Lung Cancer

10.1101/863811 ◽

2019 ◽

Author(s):

Shailender S. Chauhan ◽

Rachel K. Toth ◽

Corbin C. Jensen ◽

Andrea L. Casillas ◽

David F. Kashatus ◽

...

Keyword(s):

Lung Cancer ◽

Mitochondrial Dynamics ◽

Nsclc Patient ◽

Mitochondrial Fission ◽

Inverse Correlation ◽

Mitochondrial Superoxide ◽

Pim Kinases ◽

Response To Chemotherapy

AbstractResistance to chemotherapy represents a major obstacle to the successful treatment of non-small cell lung cancer (NSCLC). The goal of this study was to determine how PIM kinases impact mitochondrial dynamics, ROS production, and response to chemotherapy in lung cancer. Live cell imaging and microscopy were used to determine the effect of PIM loss or inhibition on mitochondrial phenotype and ROS. Inhibition of PIM kinases caused excessive mitochondrial fission and significant upregulation of mitochondrial superoxide, increasing intercellular ROS. Mechanistically, we define a signaling axis linking PIM1 to Drp1 and mitochondrial fission in lung cancer. PIM inhibition significantly increased the protein levels and mitochondrial localization of Drp1, causing marked fragmentation of mitochondria. An inverse correlation between PIM1 and Drp1 was confirmed in NSCLC patient samples. Inhibition of PIM sensitized NSCLC to chemotherapy and produced a synergistic anti-tumor response in vitro and in vivo. Immunohistochemistry and transmission electron microscopy verified that PIM inhibitors promote mitochondrial fission and apoptosis in vivo. These data improve our knowledge about how PIM1 regulates mitochondria and provide justification for combining PIM inhibition with chemotherapy in NSCLC.

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Repeated hypoglycemia blunts the responsiveness of glucose-inhibited GHRH neurons by remodeling neural inputs and disrupting mitochondrial structure and function

10.1101/788869 ◽

2019 ◽

Author(s):

M Bayne ◽

A Alvarsson ◽

K Devarakonda ◽

R Li ◽

M Jimenez-Gonzalez ◽

...

Keyword(s):

Mitochondrial Dynamics ◽

Mitochondrial Fission ◽

Glucose Deprivation ◽

Glucose Sensing ◽

Diabetic Patients ◽

Hypoglycemia Unawareness ◽

Atp Production ◽

Low Glucose

AbstractHypoglycemia is a frequent complication of diabetes, limiting therapy and increasing morbidity and mortality. With recurrent hypoglycemia, the counter-regulatory response (CRR) to decreased blood glucose is blunted, resulting in hypoglycemia unawareness. The mechanisms leading to these blunted effects remain incompletely understood. Here, we identify, with in situ hybridization, immunohistochemistry and the tissue clearing capability of iDisco, that GHRH neurons represent a unique population of arcuate nucleus neurons activated by glucose deprivation in vivo. Repeated glucose deprivation reduces GHRH neuron activation and remodels excitatory and inhibitory inputs to GHRH neurons. We show low glucose sensing is coupled to GHRH neuron depolarization, decreased ATP production and mitochondrial fusion. Repeated hypoglycemia attenuates these responses during low glucose. By maintaining mitochondrial length with the small molecule, mdivi-1, we preserved hypoglycemia sensitivity in vitro and in vivo. Our findings present possible mechanisms for the blunting of the CRR, broaden significantly our understanding of the structure of GHRH neurons and for the fist time, propose that mitochondrial dynamics play an important role in hypoglycemia unawareness. We conclude that interventions targeting mitochondrial fission in GHRH neurons may offer a new pathway to prevent hypoglycemia unawareness in diabetic patients.

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CCAAT/Enhancer-Binding Protein Homologous Protein (CHOP) Deficiency Attenuates Heatstroke-Induced Intestinal Injury

Inflammation ◽

10.1007/s10753-021-01577-x ◽

2021 ◽

Author(s):

Yan Cao ◽

Maiying Fan ◽

Yanfang Pei ◽

Lei Su ◽

Weiwei Xiao ◽

...

Keyword(s):

Protein Expression ◽

Er Stress ◽

Intestinal Barrier ◽

Intestinal Injury ◽

Intestinal Epithelial ◽

Barrier Dysfunction ◽

Homologous Protein

Abstract The intestine is one of the main target organs involved in the pathological process of heatstroke. CCAAT/enhancer-binding protein homologous protein (CHOP) is involved in endoplasmic reticulum (ER) stress-induced apoptosis. This study aimed to explore the role of CHOP in heatstroke-induced intestinal injury and potential therapy. An in vitro heat stress (HS) model using Caco-2 cells was employed. We observed the role of CHOP in apoptosis-mediated intestinal epithelial cell injury secondary to HS by evaluating cell viability, lactate dehydrogenase release, apoptosis levels, and GRP78, PERK, ATF4, CHOP, Bcl-2, and BAX mRNA and protein expression. To further study the role of CHOP in HS-induced intestinal barrier dysfunction, we assessed transepithelial electrical resistance, paracellular tracer flux, ultrastructure of tight junctions, and protein expression of ZO-1 and occludin. Male wild-type mice and CHOP knockout mice were used for in vivo experiments. We evaluated serum d-lactate and diamine oxidase levels, histopathological changes, intestinal ultrastructure, and ZO-1 and occludin protein expression. HS activated the PERK-CHOP pathway and promoted apoptosis by upregulating BAX and downregulating Bcl-2; these effects were prevented by CHOP silencing. Intestinal epithelial barrier function was disrupted by HS in vitro and in vivo. CHOP silencing prevented intestinal barrier dysfunction in Caco-2 cells, whereas CHOP knockout mice exhibited decreased intestinal mucosal injury. The ER stress inhibitor 4-phenylbutyrate (4-PBA) prevented HS-induced intestinal injury in vitro and in vivo. This study indicated that CHOP deficiency attenuates heatstroke-induced intestinal injury and may contribute to the identification of a novel therapy against heatstroke associated with the ER stress pathway.

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Streptolysin-induced endoplasmic reticulum stress promotes group A streptococcal in vivo biofilm formation and necrotizing fasciitis

10.1101/183012 ◽

2017 ◽

Author(s):

Anuradha Vajjala ◽

Debabrata Biswas ◽

Kelvin Kian Long Chong ◽

Wei Hong Tay ◽

Emanuel Hanski ◽

...

Keyword(s):

Soft Tissue ◽

Er Stress ◽

Necrotizing Fasciitis ◽

Biofilm Formation ◽

Mammalian Cells ◽

Chronic Infections ◽

Group A

AbstractGroup A Streptococcus (GAS) is a human pathogen that causes infections ranging from mild to fulminant and life-threatening. Biofilms have been implicated in acute GAS soft-tissue infections such as necrotizing fasciitis (NF). However, most in vitro models used to study GAS biofilms have been designed to mimic chronic infections and insufficiently recapitulate in vivo conditions and the host-pathogen interactions that might influence biofilm formation. Here we establish and characterize an in vitro model of GAS biofilm development on mammalian cells that simulates microcolony formation observed in a murine model of human NF. We show that on mammalian cells, GAS forms dense aggregates that display hallmark biofilm characteristics including a three-dimensional architecture and enhanced tolerance to antibiotics. In contrast to abiotic-grown biofilms, host-associated biofilms require the expression of secreted GAS streptolysins O and S (SLO, SLS) resulting in the release of a host-associated biofilm promoting-factor(s). Supernatants from GAS-infected mammalian cells or from cells treated with endoplasmic reticulum (ER) stressors restore biofilm formation to an SLO and SLS null mutant that is otherwise attenuated in biofilm formation on cells, together suggesting a role for streptolysin-induced ER stress in this process. In an in vivo mouse model, the streptolysin-null mutant is attenuated in both microcolony formation and bacterial spread, but pre-treatment of softtissue with an ER-stressor restores the ability of the mutant to form wild type like microcolonies that disseminate throughout the soft tissue. Taken together, we have identified a new role of streptolysin-driven ER stress in GAS biofilm formation and NF disease progression.Significance StatementAlthough it is well-accepted that bacterial biofilms are associated with many chronic infections, little is known about the mechanisms by which group A Streptococcus (GAS) biofilms contribute to acute soft tissue-invasive diseases like necrotizing fasciitis (NF). In this study, we establish a physiologically relevant in vitro model to study GAS biofilm formation on mammalian cells and validate our findings in a mouse model that mimics human NF. This study demonstrates a novel role of GAS streptolysin-mediated ER stress in the development and spread of GAS biofilms in acute softtissue infections. We also show that biofilm formation depends on the release of a host-associated factor that promotes microcolony formation and GAS dissemination in vivo.

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RhoGDIα-dependent balance between RhoA and RhoC is a key regulator of cancer cell tumorigenesis

Molecular Biology of the Cell ◽

10.1091/mbc.e11-01-0020 ◽

2011 ◽

Vol 22 (17) ◽

pp. 3263-3275 ◽

Cited By ~ 23

Author(s):

T. T. Giang Ho ◽

Audrey Stultiens ◽

Johanne Dubail ◽

Charles M. Lapière ◽

Betty V. Nusgens ◽

...

Keyword(s):

Tumor Growth ◽

Cancer Progression ◽

Dynamic Balance ◽

Cellular Functions ◽

Expression Of Genes ◽

Genes Encoding ◽

Interfering Rna

RhoGTPases are key signaling molecules regulating main cellular functions such as migration, proliferation, survival, and gene expression through interactions with various effectors. Within the RhoA-related subclass, RhoA and RhoC contribute to several steps of tumor growth, and the regulation of their expression affects cancer progression. Our aim is to investigate their respective contributions to the acquisition of an invasive phenotype by using models of reduced or forced expression. The silencing of RhoC, but not of RhoA, increased the expression of genes encoding tumor suppressors, such as nonsteroidal anti-inflammatory drug–activated gene 1 (NAG-1), and decreased migration and the anchorage-independent growth in vitro. In vivo, RhoC small interfering RNA (siRhoC) impaired tumor growth. Of interest, the simultaneous knockdown of RhoC and NAG-1 repressed most of the siRhoC-related effects, demonstrating the central role of NAG-1. In addition of being induced by RhoC silencing, NAG-1 was also largely up-regulated in cells overexpressing RhoA. The silencing of RhoGDP dissociation inhibitor α (RhoGDIα) and the overexpression of a RhoA mutant unable to bind RhoGDIα suggested that the effect of RhoC silencing is indirect and results from the up-regulation of the RhoA level through competition for RhoGDIα. This study demonstrates the dynamic balance inside the RhoGTPase network and illustrates its biological relevance in cancer progression.

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