Organelle isolation: functional mitochondria from mouse liver, muscle and cultured filroblasts

Frezza, Christian; Cipolat, Sara; Scorrano, Luca

doi:10.1038/nprot.2006.478

Cited by 1,025 publications

(863 citation statements)

References 25 publications

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“…For O 2 consumption and electron microscopy experiments, mitochondria were purified as described. 52 Mitochondrial pellet was resuspended in respiration buffer (150 mM KCl, 10 mM Tris/MOPS and 10 μM ATP). Mitochondria (400 μg) were transferred to a Clark electrode chamber to measure oxygen consumption.…”

Section: Discussionmentioning

confidence: 99%

Granzyme B-induced mitochondrial ROS are required for apoptosis

et al. 2014

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Caspases and the cytotoxic lymphocyte protease granzyme B (GB) induce reactive oxygen species (ROS) formation, loss of transmembrane potential and mitochondrial outer membrane permeabilization (MOMP). Whether ROS are required for GBmediated apoptosis and how GB induces ROS is unclear. Here, we found that GB induces cell death in an ROS-dependent manner, independently of caspases and MOMP. GB triggers ROS increase in target cell by directly attacking the mitochondria to cleave NDUFV1, NDUFS1 and NDUFS2 subunits of the NADH: ubiquinone oxidoreductase complex I inside mitochondria. This leads to mitocentric ROS production, loss of complex I and III activity, disorganization of the respiratory chain, impaired mitochondrial respiration and loss of the mitochondrial cristae junctions. Furthermore, we have also found that GB-induced mitocentric ROS are necessary for optimal apoptogenic factor release, rapid DNA fragmentation and lysosomal rupture. Interestingly, scavenging the ROS delays and reduces many of the features of GB-induced death. Consequently, GB-induced ROS significantly promote apoptosis. Cell Death and Differentiation (2015) 22, 862-874; doi:10.1038/cdd.2014.180; published online 31 October 2014To induce cell death, human granzyme B (GB) activates effector caspase-3 or acts directly on key caspase substrates, such as the proapoptotic BH3 only Bcl-2 family member Bid, inhibitor of caspase-activated DNase (ICAD), poly-(ADPribose) polymerase-1 (PARP-1), lamin B, nuclear mitotic apparatus protein 1 (NUMA1), catalytic subunit of the DNAdependent protein kinase (DNA-PKcs) and tubulin. [1][2][3] Consequently, caspase inhibitors have little effect on human GB-mediated cell death and DNA fragmentation. 2 GB causes reactive oxygen species (ROS) production, dissipation of the mitochondrial transmembrane potential (ΔΨm) and MOMP, which leads to the release of apoptogenic factors such as cytochrome c (Cyt c), HtrA2/Omi, endonuclease G (Endo G), Smac/Diablo and apoptosis-inducing factor, from the mitochondrial intermembrane space to the cytosol. [4][5][6][7][8][9][10][11] Interestingly, cells deficient for Bid, Bax and Bak are still sensitive to human GB-induced cell death, 5,11-13 suggesting that human GB targets the mitochondria in another way that needs to be characterized. Altogether, much attention has been focused on the importance of MOMP in the execution of GB-mediated cell death, leaving unclear whether ROS production is a bystander effect or essential to the execution of GBinduced apoptosis. The mitochondrial NADH: ubiquinone oxidoreductase complex I is a key determinant in steadystate ROS production. This 1 MDa complex, composed of 44 subunits, 14 couples the transfer of two electrons from NADH to ubiquinone with the translocation of four protons to generate the ΔΨm. The importance of ROS has been previously demonstrated for caspase-3 and granzyme A (GA) pathways through the cleavage of NDUFS1 and NDUFS3, respectively. [15][16][17][18] GA induces cell death in a Bcl-2-insensitive and caspase-and MOMP-indepen...

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Section: Discussionmentioning

confidence: 99%

Granzyme B-induced mitochondrial ROS are required for apoptosis

et al. 2014

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“…Protein extracts were isolated and quantified as previously described (Boutant et al , 2015), except for cellular fractionations studies (Frezza et al , 2007). For Western blotting, proteins were separated by SDS–PAGE and transferred onto nitrocellulose membranes.…”

Section: Methodsmentioning

confidence: 99%

Mfn2 is critical for brown adipose tissue thermogenic function

et al. 2017

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Mitochondrial fusion and fission events, collectively known as mitochondrial dynamics, act as quality control mechanisms to ensure mitochondrial function and fine‐tune cellular bioenergetics. Defective mitofusin 2 (Mfn2) expression and enhanced mitochondrial fission in skeletal muscle are hallmarks of insulin‐resistant states. Interestingly, Mfn2 is highly expressed in brown adipose tissue (BAT), yet its role remains unexplored. Using adipose‐specific Mfn2 knockout (Mfn2‐adKO) mice, we demonstrate that Mfn2, but not Mfn1, deficiency in BAT leads to a profound BAT dysfunction, associated with impaired respiratory capacity and a blunted response to adrenergic stimuli. Importantly, Mfn2 directly interacts with perilipin 1, facilitating the interaction between the mitochondria and the lipid droplet in response to adrenergic stimulation. Surprisingly, Mfn2‐adKO mice were protected from high‐fat diet‐induced insulin resistance and hepatic steatosis. Altogether, these results demonstrate that Mfn2 is a mediator of mitochondria to lipid droplet interactions, influencing lipolytic processes and whole‐body energy homeostasis.

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“…Mitochondrial complex I, IV and V activity and mitochondrial ATP concentration Muscle mitochondria were isolated according to a previously described protocol with some modifications (Frezza et al, 2007). A total of 100μg of frozen muscle sample was homogenized in a Dounce homogenizer with 20 strokes using isolation buffer 1 (w : v = 1 : 5) containing 1 M Tris/ HCL, 0.1 M EGTA/Tris, 10% BSA and 1 M sucrose; a protease inhibitor cocktail (11697498001; Roche) was also added to the isolation buffer.…”

Section: Ethics Statementmentioning

confidence: 99%

Lipopolysaccharide markedly changes glucose metabolism and mitochondrial function in the longissimus muscle of pigs

Sun

Huang

Yin

et al. 2016

Animal

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Most previous studies on the effects of lipopolysaccharide (LPS) in pigs focused on the body's immune response, and few reports paid attention to body metabolism changes. To better understand the glucose metabolism changes in skeletal muscle following LPS challenge and to clarify the possible mechanism, 12 growing pigs were employed. Animals were treated with either 2 ml of saline or 15 µg/kg BW LPS, and samples were collected 6 h later. The glycolysis status and mitochondrial function in the longissimus dorsi (LD) muscle of pigs were analyzed. The results showed that serum lactate content and NADH content in LD muscle significantly increased compared with the control group. Most glycolysis-related genes expression, as well as hexokinase, pyruvate kinase and lactic dehydrogenase activity, in LD muscle was significantly higher compared with the control group. Mitochondrial complexes I and IV significantly increased, while mitochondrial ATP concentration markedly decreased. Significantly increased calcium content in the mitochondria was observed, and endoplasm reticulum (ER) stress has been demonstrated in the present study. The results showed that LPS treatment markedly changes glucose metabolism and mitochondrial function in the LD muscle of pigs, and increased calcium content induced by ER stress was possibly involved. The results provide new clues for clarifying metabolic diseases in muscle induced by LPS.Keywords: lipopolysaccharide, mitochondria, glycolysis, pig, inflammation ImplicationsSkeletal muscle is the primary tissue responsible for wholebody energy metabolism. In the current study, we designed the experiment to investigate the effect of lipopolysaccharide (LPS) treatment on muscle energy metabolism in pigs. The results indicated that LPS treatment significantly changed glycolysis level and mitochondrial function in the longissimus dorsi (LD) muscle of pigs, and increased calcium content induced by endoplasm reticulum stress was possibly involved. Understanding these underlying mechanisms may provide new therapeutic strategies for treating metabolic diseases induced by LPS in muscle.

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Organelle isolation: functional mitochondria from mouse liver, muscle and cultured filroblasts

Cited by 1,025 publications

References 25 publications

Granzyme B-induced mitochondrial ROS are required for apoptosis

Granzyme B-induced mitochondrial ROS are required for apoptosis

Mfn2 is critical for brown adipose tissue thermogenic function

Lipopolysaccharide markedly changes glucose metabolism and mitochondrial function in the longissimus muscle of pigs

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