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.

Chemosensitization by knockdown of adenine nucleotide translocase-2.

Mitochondrial membrane permeabilization (MMP) is a rate-limiting step of apoptosis, including in anticancer chemotherapy. Adenine nucleotide translocase (ANT) mediates the exchange of ADP and ATP on the inner mitochondrial membrane in healthy cells. In addition, ANT can cooperate with Bax to form a lethal pore during apoptosis. Humans possess four distinct ANT isoforms, encoded by four genes, whose transcription depends on the cell type, developmental stage, cell proliferation, and hormone status. Here, we show that the ANT2 gene is up-regulated in several hormone-dependent cancers. Knockdown of ANT2 by RNA interference induced no major changes in the aspect of the mitochondrial network or cell cycle but provoked minor increase in mitochondrial transmembrane potential and reactive oxygen species level and reduced intracellular ATP concentration without affecting glycolysis. At expression and functional levels, ANT2 depletion was not compensated by other ANT isoforms. Most importantly, ANT2, but not ANT1, silencing facilitated MMP induction by lonidamine, a mitochondrion-targeted antitumor compound already used in clinical studies for breast, ovarian, glioma, and lung cancer as well as prostate adenoma. The combination of ANT2 knockdown with lonidamine induced apoptosis irrespective of the Bcl-2 status. These data identify ANT2 as an endogenous inhibitor of MMP and suggest that its selective inhibition could constitute a promising strategy of chemosensitization.

Therapeutic peptides: Targeting the mitochondrion to modulate apoptosis.

For many years, medical drug discovery has extensively exploited peptides as lead compounds. Currently, novel structures of therapeutic peptides are derived from active pre-existing peptides or from high-throughput screening, and optimized following a rational drug design approach. Molecules of interest may prove their ability to influence the disease outcome in animal models and must respond to a set of criteria based on toxicity studies, ease of administration, the cost of their synthesis, and logistic for clinical use to validate it as a good candidate in a therapeutic perspective. This applies to the potential use of peptides to target one central intracellular organelle, the mitochondrion, to modulate (i.e. activate or prevent) apoptosis. Putative mitochondrial protein targets and the strategies already elaborated to correct the defects linked to these proteins (overexpression, inactivation, mutation..., etc.) are described, and recent advances that led or may lead to the conception of therapeutic peptides via a specific action on these mitochondrial targets in the future are discussed.