Respiration and substrate transport rates as well as reactive oxygen species production distinguish mitochondria from brain and liver

Events defining the progression to human type 1 diabetes (T1D) have remained elusive owing to the complex interaction between genetics, the immune system, and the environment. Type 1 interferons (T1-IFN) are known to be a constituent of the autoinflammatory milieu within the pancreas of patients with T1D. However, the capacity of IFNα/β to modulate human activated autoreactive CD8+ T-cell (cytotoxic T lymphocyte) responses within the islets of patients with T1D has not been investigated. Here, we engineer human β-cell–specific cytotoxic T lymphocytes and demonstrate that T1-IFN augments cytotoxicity by inducing rapid phosphorylation of STAT4, resulting in direct binding at the granzyme B promoter within 2 h of exposure. The current findings provide novel insights concerning the regulation of effector function by T1-IFN in human antigen-experienced CD8+ T cells and provide a mechanism by which the presence of T1-IFN potentiates diabetogenicity within the autoimmune islet.

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“…Western blots were performed as previously described ( 38 ). Proteins were resolved on 4–20% SDS-PAGE gels.…”

Section: Methodsmentioning

confidence: 99%

Type 1 Interferons Potentiate Human CD8+ T-Cell Cytotoxicity Through a STAT4- and Granzyme B–Dependent Pathway

et al. 2017

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“…Before drug treatment, the protein concentration was adjusted to 1 mg/ml using appropriate buffer. The purity of mitochondria was ascertained by detecting four mitochondrion-specific proteins and the cytosolic protein glyceraldehyde-3-phosphate dehydrogenase (GAPDH) using Western blot (Gusdon et al 2015 ). Briefly, mitochondrial and cytosolic fractions from four different preparations using four rats were diluted using the Laemmli protein loading buffer and subject to SDS-PAGE with 4–20% gradient gels.…”

Section: Methodsmentioning

confidence: 99%

Effects of 31 FDA approved small-molecule kinase inhibitors on isolated rat liver mitochondria

et al. 2016

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The FDA has approved 31 small-molecule kinase inhibitors (KIs) for human use as of November 2016, with six having black box warnings for hepatotoxicity (BBW-H) in product labeling. The precise mechanisms and risk factors for KI-induced hepatotoxicity are poorly understood. Here, the 31 KIs were tested in isolated rat liver mitochondria, an in vitro system recently proposed to be a useful tool to predict drug-induced hepatotoxicity in humans. The KIs were incubated with mitochondria or submitochondrial particles at concentrations ranging from therapeutic maximal blood concentrations (Cmax) levels to 100-fold Cmax levels. Ten endpoints were measured, including oxygen consumption rate, inner membrane potential, cytochrome c release, swelling, reactive oxygen species, and individual respiratory chain complex (I–V) activities. Of the 31 KIs examined only three including sorafenib, regorafenib and pazopanib, all of which are hepatotoxic, caused significant mitochondrial toxicity at concentrations equal to the Cmax, indicating that mitochondrial toxicity likely contributes to the pathogenesis of hepatotoxicity associated with these KIs. At concentrations equal to 100-fold Cmax, 18 KIs were found to be toxic to mitochondria, and among six KIs with BBW-H, mitochondrial injury was induced by regorafenib, lapatinib, idelalisib, and pazopanib, but not ponatinib, or sunitinib. Mitochondrial liability at 100-fold Cmax had a positive predictive power (PPV) of 72% and negative predictive power (NPV) of 33% in predicting human KI hepatotoxicity as defined by product labeling, with the sensitivity and specificity being 62% and 44%, respectively. Similar predictive power was obtained using the criterion of Cmax ≥1.1 µM or daily dose ≥100 mg. Mitochondrial liability at 1–2.5-fold Cmax showed a 100% PPV and specificity, though the NPV and sensitivity were 32% and 14%, respectively. These data provide novel mechanistic insights into KI hepatotoxicity and indicate that mitochondrial toxicity at therapeutic levels can help identify hepatotoxic KIs.Electronic supplementary materialThe online version of this article (doi:10.1007/s00204-016-1918-1) contains supplementary material, which is available to authorized users.

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“…Expression of total c-Jun N-terminal kinases (JNK), phospho-JNK (p-JNK), total P38 and phospho-P38 (p-P38) in liver were measured by Western blotting as described [25]. Proteins were separated on 12.5% SDS-PAGE gels and transferred onto nitrocellulose membranes.…”

Section: Methodsmentioning

confidence: 99%

Melatonin improves non-alcoholic fatty liver disease via MAPK-JNK/P38 signaling in high-fat-diet-induced obese mice

et al. 2016

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BackgroundMelatonin can regulate lipid metabolism, increase insulin sensitivity, regulate glucose metabolism and reduce body weight. This study is aimed to determine the effects and mechanism of action of melatonin on non-alcoholic fatty liver disease (NAFLD) in high-fat-diet (HFD) induced obese mice.MethodsNAFLD was induced by HFD in C57BL/6 mice. A total of 24 mice were randomly assigned to 4 groups. Groups A and B were fed with HFD for 36 weeks while groups C and D were fed with a regular diet (RD). During the last 12 weeks, Groups A and C were treated with 10 mg/kg melatonin while Groups B and D were treated with water in the same volume by intragastric administration. Body and liver weight, blood glucose, serum transaminases and lipid levels, and markers of hepatic inflammation were measured. Histological analyses were also performed on liver tissue.ResultsAfter 12 weeks of treatment with melatonin, body weights (Group A: before 53.11 ± 0.72 vs after 12w treatment 39.48 ± 0.74) and liver weights (A 1.93 ± 0.09 g vs B 2.92 ± 0.19 g vs C 1.48 ± 0.09 g vs D 1.49 ± 0.10 g), fasting plasma glucose, alanine transaminase (A 24.33 ± 11.90 IU/L vs B 60.80 ± 10.18 IU/L vs C 13.01 ± 3.49 IU/L vs D 16.62 ± 2.00 IU/L), and low-density cholesterol (A 0.24 ± 0.06 mmol/L vs B 1.57 ± 0.10 mmol/L vs C 0.28 ± 0.06 mmol/L vs D 0.29 ± 0.03 mmol/L) were significantly decreased in HFD mice. HFD fed mice treated with melatonin showed significantly less liver steatosis. Treatment of HFD fed mice with melatonin led to a significant decrease in the expression of TNF-α, IL-1β, and IL-6 measured using quantitative real-time polymerase chain reaction (qRT-PCR). HFD fed mice demonstrated increased phosphorylation of P38 and JNK1/2, which was reduced by melatonin treatment.ConclusionsThe study concluded that melatonin could improve NAFLD by decreasing body weight and reduce inflammation in HFD induced obese mice by modulating the MAPK-JNK/P38 signaling pathway.

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Respiration and substrate transport rates as well as reactive oxygen species production distinguish mitochondria from brain and liver

Cited by 20 publications

References 81 publications

Type 1 Interferons Potentiate Human CD8+ T-Cell Cytotoxicity Through a STAT4- and Granzyme B–Dependent Pathway

Type 1 Interferons Potentiate Human CD8+ T-Cell Cytotoxicity Through a STAT4- and Granzyme B–Dependent Pathway

Effects of 31 FDA approved small-molecule kinase inhibitors on isolated rat liver mitochondria

Melatonin improves non-alcoholic fatty liver disease via MAPK-JNK/P38 signaling in high-fat-diet-induced obese mice

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