Adipose tissue senescence is mediated by increased ATP content after a short-term high-fat diet exposure.

In the context of obesity, senescent cells accumulate in white adipose tissue (WAT). The cellular underpinnings of WAT senescence leading to insulin resistance are not fully elucidated. The objective of the current study was to evaluate the presence of WAT senescence early after initiation of high-fat diet (HFD, 1-10 weeks) in 5-month-old male C57BL/6J mice and the potential role of energy metabolism. We first showed that WAT senescence occurred 2 weeks after HFD as evidenced in whole WAT by increased senescence-associated ß-galactosidase activity and cyclin-dependent kinase inhibitor 1A and 2A expression. WAT senescence affected various WAT cell populations, including preadipocytes, adipose tissue progenitors, and immune cells, together with adipocytes. WAT senescence was associated with higher glycolytic and mitochondrial activity leading to enhanced ATP content in HFD-derived preadipocytes, as compared with chow diet-derived preadipocytes. One-month daily exercise, introduced 5 weeks after HFD, was an effective senostatic strategy, since it reversed WAT cellular senescence, while reducing glycolysis and production of ATP. Interestingly, the beneficial effect of exercise was independent of body weight and fat mass loss. We demonstrated that WAT cellular senescence is one of the earliest events occurring after HFD initiation and is intimately linked to the metabolic state of the cells. Our data uncover a critical role for HFD-induced elevated ATP as a local danger signal inducing WAT senescence. Exercise exerts beneficial effects on adipose tissue bioenergetics in obesity, reversing cellular senescence, and metabolic abnormalities.

Drug-induced hepatic steatosis in absence of severe mitochondrial dysfunction in HepaRG cells: proof of multiple mechanism-based toxicity.

Steatosis is a liver lesion reported with numerous pharmaceuticals. Prior studies showed that severe impairment of mitochondrial fatty acid oxidation (mtFAO) constantly leads to lipid accretion in liver. However, much less is known about the mechanism(s) of drug-induced steatosis in the absence of severe mitochondrial dysfunction, although previous studies suggested the involvement of mild-to-moderate inhibition of mtFAO, increased de novo lipogenesis (DNL), and impairment of very low-density lipoprotein (VLDL) secretion. The objective of our study, mainly carried out in human hepatoma HepaRG cells, was to investigate these 3 mechanisms with 12 drugs able to induce steatosis in human: amiodarone (AMIO, used as positive control), allopurinol (ALLO), D-penicillamine (DPEN), 5-fluorouracil (5FU), indinavir (INDI), indomethacin (INDO), methimazole (METHI), methotrexate (METHO), nifedipine (NIF), rifampicin (RIF), sulindac (SUL), and troglitazone (TRO). Hepatic cells were exposed to drugs for 4 days with concentrations decreasing ATP level by less than 30% as compared to control and not exceeding 100 × Cmax. Among the 12 drugs, AMIO, ALLO, 5FU, INDI, INDO, METHO, RIF, SUL, and TRO induced steatosis in HepaRG cells. AMIO, INDO, and RIF decreased mtFAO. AMIO, INDO, and SUL enhanced DNL. ALLO, 5FU, INDI, INDO, SUL, RIF, and TRO impaired VLDL secretion. These seven drugs reduced the mRNA level of genes playing a major role in VLDL assembly and also induced endoplasmic reticulum (ER) stress. Thus, in the absence of severe mitochondrial dysfunction, drug-induced steatosis can be triggered by different mechanisms, although impairment of VLDL secretion seems more frequently involved, possibly as a consequence of ER stress.

Use of human cancer cell lines mitochondria to explore the mechanisms of BH3 peptides and ABT-737-induced mitochondrial membrane permeabilization.

Current limitations of chemotherapy include toxicity on healthy tissues and multidrug resistance of malignant cells. A number of recent anti-cancer strategies aim at targeting the mitochondrial apoptotic machinery to induce tumor cell death. In this study, we set up protocols to purify functional mitochondria from various human cell lines to analyze the effect of peptidic and xenobiotic compounds described to harbour either Bcl-2 inhibition properties or toxic effects related to mitochondria. Mitochondrial inner and outer membrane permeabilization were systematically investigated in cancer cell mitochondria versus non-cancerous mitochondria. The truncated (t-) Bid protein, synthetic BH3 peptides from Bim and Bak, and the small molecule ABT-737 induced a tumor-specific and OMP-restricted mitochondrio-toxicity, while compounds like HA-14.1, YC-137, Chelerythrine, Gossypol, TW-37 or EM20-25 did not. We found that ABT-737 can induce the Bax-dependent release of apoptotic proteins (cytochrome c, Smac/Diablo and Omi/HtrA2 but not AIF) from various but not all cancer cell mitochondria. Furthermore, ABT-737 addition to isolated cancer cell mitochondria induced oligomerization of Bax and/or Bak monomers already inserted in the mitochondrial membrane. Finally immunoprecipatations indicated that ABT-737 induces Bax, Bak and Bim desequestration from Bcl-2 and Bcl-xL but not from Mcl-1L. This study investigates for the first time the mechanism of action of ABT-737 as a single agent on isolated cancer cell mitochondria. Hence, this method based on MOMP (mitochondrial outer membrane permeabilization) is an interesting screening tool, tailored for identifying Bcl-2 antagonists with selective toxicity profile against cancer cell mitochondria but devoid of toxicity against healthy mitochondria.

The fourth isoform of the adenine nucleotide translocator inhibits mitochondrial apoptosis in cancer cells.

The adenine nucleotide translocator (ANT) is a mitochondrial bi-functional protein, which catalyzes the exchange of ADP and ATP between cytosol and mitochondria and participates in many models of mitochondrial apoptosis. The human adenine nucleotide translocator sub-family is composed of four isoforms, namely ANT1-4, encoded by four nuclear genes, whose expression is highly regulated. Previous studies have revealed that ANT1 and 3 induce mitochondrial apoptosis, whereas ANT2 is anti-apoptotic. However, the role of the recently identified isoform ANT4 in the apoptotic pathway has not yet been elucidated. Here, we investigated the effects of stable heterologous expression of the ANT4 on proliferation, mitochondrial respiration and cell death in human cancer cells, using ANT3 as a control of pro-apoptotic isoform. As expected, ANT3 enhanced mitochondria-mediated apoptosis in response to lonidamine, a mitochondriotoxic chemotherapeutic drug, and staurosporine, a protein kinase inhibitor. Our results also indicate that the pro-apoptotic effect of ANT3 was accompanied by decreased rate of cell proliferation, alteration in the mitochondrial network topology, and decreased reactive oxygen species production. Of note, we demonstrate for the first time that ANT4 enhanced cell growth without impacting mitochondrial network or respiration. Moreover, ANT4 differentially regulated the intracellular levels of hydrogen peroxide without affecting superoxide anion levels. Finally, stable ANT4 overexpression protected cancer cells from lonidamine and staurosporine apoptosis in a manner independent of Bcl-2 expression. These data highlight a hitherto undefined cytoprotective activity of ANT4, and provide a novel dichotomy in the human ANT isoform sub-family with ANT1 and 3 isoforms functioning as pro-apoptotic while ANT2 and 4 isoforms render cells resistant to death inducing stimuli.

A flavivirus protein M-derived peptide directly permeabilizes mitochondrial membranes, triggers cell death and reduces human tumor growth in nude mice.

Dengue viruses belong to the Flavivirus family and are responsible for hemorrhagic fever in Human. Dengue virus infection triggers apoptosis especially through the expression of the small membrane (M) protein. Using isolated mitochondria, we found that synthetic peptides containing the C-terminus part of the M ectodomain caused apoptosis-related mitochondrial membrane permeabilization (MMP) events. These events include matrix swelling and the dissipation of the mitochondrial transmembrane potential (DeltaPsi(m)). Protein M Flavivirus sequence alignments and helical wheel projections reveal a conserved distribution of charged residues. Moreover, when combined to the cell penetrating HIV-1 Tat peptide transduction domain (Tat-PTD), this sequence triggers a caspase-dependent cell death associated with DeltaPsi(m) loss and cytochrome c release. Mutational approaches coupled to functional screening on isolated mitochondria resulted in the selection of a protein M derived sequence containing nine residues with potent MMP-inducing properties on isolated mitochondria. A chimeric peptide composed of a Tat-PTD linked to the 9-mer entity triggers MMP and cell death. Finally, local administration of this chimeric peptide induces growth inhibition of xenograft prostate PC3 tumors in immuno-compromised mice, and significantly enhances animal survival. Together, these findings support the notion of using viral genomes as valuable sources to discover mitochondria-targeted sequences that may lead to the development of new anticancer compounds.

Trefoil factor TFF1-induced protection of conjunctival cells from apoptosis at premitochondrial and postmitochondrial levels.

PURPOSE: Goblet cells of the conjunctival epithelium synthesize and secrete TFF1 (Trefoil factor 1), a small protease-resistant peptide that, together with mucins, is responsible for the rheologic properties of the tear film. This study aimed to determine whether TFF1, whose synthesis increases in inflammatory conditions such as pterygium, could protect conjunctival cells from apoptosis. METHODS: Chang conjunctival cells, either wild-type or expressing TFF1 through stable transfection, were exposed to benzalkonium chloride (BAK) and ultraviolet (UV) irradiation to trigger apoptosis. The authors used cell fractionation to detect lipid raft-associated proteins, coimmunoprecipitation to explore the formation of a death-inducing signaling complex (DISC), and a combination of immunofluorescence, immunoblotting, flow cytometry, siRNA-mediated decrease in gene expression, and electrophoretic mobility shift assay to explore the mechanisms of TFF1-protective effects. RESULTS: TFF1 protects Chang conjunctival cells from apoptosis induced by UV irradiation and BAK at two levels. First, TFF1 prevents caspase-8 activation at the level of the DISC that involves Fas receptor in plasma membrane rafts, which in turn decreases the mitochondrial release of cytochrome c. Second, TFF1 interferes with caspase-9 and caspase-3 activation through an NF-kappaB-induced increase in the expression of XIAP (X-linked inhibitor of apoptosis protein). CONCLUSIONS: TFF1 upregulation on inflammatory conditions may be a protective mechanism that limits conjunctival cell loss by inhibiting apoptosis upstream and downstream of the mitochondrial events. These observations suggest a potential interest of TFF1 or related peptides to prevent cell death in ocular surface disorders.

Differential mechanisms of conjunctival cell death induction by ultraviolet irradiation and benzalkonium chloride.

PURPOSE: To determine the molecular mechanisms of conjunctival cell death on exposure to the quaternary ammonium preservative benzalkonium chloride (BAC) and ultraviolet (UV) irradiation. METHODS: Chang conjunctival cells, either wild-type or stably transfected with various constructs encoding antiapoptotic molecules or transiently transfected with siRNA targeting the beclin-1 gene, were exposed to BAC or UV radiation Cell death was analyzed morphologically with fluorescence and electron microscopy, and molecular mechanisms of death were studied by using immunofluorescence, cell fractionation, caspase substrates, and immunoblot analysis, with or without immunoprecipitation. The main results were controlled in IOBA-NHC cells. RESULTS: Both agents induced cytochrome c release from the mitochondria, caspase activation, and nuclear chromatin condensation, suggesting caspase-dependent apoptosis. These events are prevented by stable expression of Bcl-2 protein. Both agents also induced a redistribution of Fas in plasma membrane rafts and the Fas-ligand-independent formation of a death-inducing complex leading to caspase-8 activation. Stable expression of either a dominant negative construct of Fas-associated death domain (FADD) or the long or short isoform of FADD-like interleukin-1-beta-converting enzyme inhibitory protein (FLIP) inhibited caspase-8 activation in response to both UV radiation and BAC. However, these proteins, as well as permeant peptides and baculovirus p35 caspase-inhibitors, delayed more efficiently the UV irradiation-induced than the BAC-induced nuclear chromatin condensation. BAC specifically activated a caspase-independent pathway by inducing the mitochondrial release of apoptosis-inducing factor. BAC-treated cells contain autophagosomes/autolysosomes, a characteristic feature of autophagy, and siRNA-mediated downregulation of the beclin-1 gene, whose product is crucial for autophagy, increases BAC toxicity. CONCLUSIONS: UV irradiation induces typical, caspase-dependent cell death, whereas death induced by BAC associates features of caspase-dependent and -independent apoptosis counteracted by an autophagic process.

Trefoil factor family mRNA and protein expression in pterygium.

Trefoil factor family (TFF) of proteins are involved in mucosal protection and healing and are induced in inflammatory diseases and neoplastic progression. The purpose of this investigation was to determine if expression of the trefoil factor family (TFF) proteins is altered in human pterygium compared to in normal conjunctiva. Fourteen pterygia (P) and 21 biopsies from normal human conjunctiva (NC) were studied. TFF1, TFF2 and TFF3 mRNA levels were measured by semi-quantitative reverse-transcription polymerase chain reaction (RT-PCR), and TFF1 mRNA levels in addition by real-time PCR. The cellular expression of TFF1 (pS2), TFF3 (intestinal trefoil factor) and M1/MUC5AC mucin in ten pterygia and ten normal human conjunctiva specimens was analyzed by immunohistochemistry using specific monoclonal antibodies. TFF1 mRNA levels were higher in P than in NC (p=0.02). Accordingly, intensity of TFF1 and mucin MUC5AC immunostaining was higher in P than in NC. Mucus-secreting goblet cells (GC) were more densely packed in P than in NC. In both cases, TFF1 protein was detected in GC only, but was not systematically expressed in all GC. In addition, TFF3 mRNA levels were similar (p=0.89) in NC and P, while TFF2 (spasmolytic polypeptide) mRNA were not detected. Both TFF3 and MUC5AC proteins were clearly detected in all GC identified in NC and P. Increased expression of TFF1 mRNA and protein is observed in pterygium GC, suggesting that this trefoil protein might exert protective and beneficial roles during the pathogenesis of this benign and inflammatory conjunctival tumor.