Explore the vast range of titles available.

Chronic inflammation is a common feature of obesity, with elevated cytokines such as interleukin-1 (IL-1) in the circulation and tissues. Here, we report an unconventional IL-1R–MyD88–IRAK2–PHB/OPA1 signaling axis that reprograms mitochondrial metabolism in adipocytes to exacerbate obesity. IL-1 induced recruitment of IRAK2 Myddosome to mitochondria outer membranes via recognition by TOM20, followed by TIMM50-guided translocation of IRAK2 into mitochondria inner membranes, to suppress oxidative phosphorylation and fatty acid oxidation, thereby attenuating energy expenditure. Adipocyte-specific MyD88 or IRAK2 deficiency reduced high-fat-diet-induced weight gain, increased energy expenditure and ameliorated insulin resistance, associated with a smaller adipocyte size and increased cristae formation. IRAK2 kinase inactivation also reduced high-fat diet-induced metabolic diseases. Mechanistically, IRAK2 suppressed respiratory super-complex formation via interaction with PHB1 and OPA1 upon stimulation of IL-1. Taken together, our results suggest that the IRAK2 Myddosome functions as a critical link between inflammation and metabolism, representing a novel therapeutic target for patients with obesity. Obesity is often accompanied by chronic inflammation. Li and colleagues show that, in mice fed high-fat diets, IL-1 signaling in adipocytes induces an unconventional IRAK2 translocation to mitochondria and suppresses respiratory super-complex formation to alter mitochondrial function, and exacerbates obesity.

Metabolic dysfunction accompanies neurodegenerative disease and aging. An important step for therapeutic development is a more sophisticated understanding of the source of metabolic dysfunction, as well as to distinguish disease-associated changes from aging effects. We examined mitochondrial function in ex vivo aging and glaucomatous optic nerve using a novel approach, the Seahorse Analyzer. Optic nerves (ON) from the DBA/2J mouse model of glaucoma and the DBA/2- Gpnmb + control strain were isolated, and oxygen consumption rate (OCR) and extracellular acidification rate (ECAR), the discharge of protons from lactate release or byproducts of substrate oxidation, were measured. The glial-specific aconitase inhibitor fluorocitrate was used to limit the contribution of glial mitochondria to OCR and ECAR. We observed significant decreases in maximal respiration, ATP production, and spare capacity with aging. In the presence of fluorocitrate, OCR was higher, with more ATP produced, in glaucoma compared to aged ON. However, glaucoma ON showed lower maximal respiration. In the presence of fluorocitrate and challenged with ATPase inhibition, glaucoma ON was incapable of further upregulation of glycolysis to compensate for the loss of oxidative phosphorylation. Inclusion of 2-deoxyglucose as a substrate during ATPase inhibition indicated a significantly higher proportion of ECAR was derived from TCA cycle substrate oxidation than glycolysis in glaucoma ON. These data indicate that glaucoma axons have limited ability to respond to increased energy demand given their lower maximal respiration and inability to upregulate glycolysis when challenged. The higher ATP output from axonal mitochondria in glaucoma optic nerve compensates for this lack of resiliency but is ultimately inadequate for continued function.

The mitochondrial electrochemical gradient (Δ p ), which comprises the pH gradient (ΔpH) and the membrane potential (ΔΨ), is crucial in controlling energy transduction. During myocardial ischemia and reperfusion (IR), mitochondrial dysfunction mediates superoxide ( · O 2 − ) and H 2 O 2 overproduction leading to oxidative injury. However, the role of ΔpH and ΔΨ in post-ischemic injury is not fully established. Here we studied mitochondria from the risk region of rat hearts subjected to 30 min of coronary ligation and 24 h of reperfusion in vivo. In the presence of glutamate, malate and ADP, normal mitochondria (mitochondria of non-ischemic region, NR) exhibited a heightened state 3 oxygen consumption rate (OCR) and reduced · O 2 − and H 2 O 2 production when compared to state 2 conditions. Oligomycin (increases ΔpH by inhibiting ATP synthase) increased · O 2 − and H 2 O 2 production in normal mitochondria, but not significantly in the mitochondria of the risk region (IR mitochondria or post-ischemic mitochondria), indicating that normal mitochondrial · O 2 − and H 2 O 2 generation is dependent on ΔpH and that IR impaired the ΔpH of normal mitochondria. Conversely, nigericin (dissipates ΔpH) dramatically reduced · O 2 − and H 2 O 2 generation by normal mitochondria under state 4 conditions, and this nigericin quenching effect was less pronounced in IR mitochondria. Nigericin also increased mitochondrial OCR, and predisposed normal mitochondria to a more oxidized redox status assessed by increased oxidation of cyclic hydroxylamine, CM-H. IR mitochondria, although more oxidized than normal mitochondria, were not responsive to nigericin-induced CM-H oxidation, which is consistent with the result that IR induced ΔpH impairment in normal mitochondria. Valinomycin, a K + ionophore used to dissipate ΔΨ, drastically diminished · O 2 − and H 2 O 2 generation by normal mitochondria, but less pronounced effect on IR mitochondria under state 4 conditions, indicating that ΔΨ also contributed to · O 2 − generation by normal mitochondria and that IR mediated ΔΨ impairment. However, there was no significant difference in valinomycin-induced CM-H oxidation between normal and IR mitochondria. In conclusion, under normal conditions the proton backpressure imposed by ΔpH restricts electron flow, controls a limited amount of · O 2 − generation, and results in a more reduced myocardium; however, IR causes ΔpH impairment and prompts a more oxidized myocardium.

We demonstrated previously that TRPV1-dependent coupling of coronary blood flow (CBF) to metabolism is disrupted in diabetes. A critical amount of H 2 O 2 contributes to CBF regulation; however, excessive H 2 O 2 impairs responses. We sought to determine the extent to which differential regulation of TRPV1 by H 2 O 2 modulates CBF and vascular reactivity in diabetes. We used contrast echocardiography to study TRPV1 knockout (V1KO), db/db diabetic, and wild type C57BKS/J (WT) mice. H 2 O 2 dose-dependently increased CBF in WT mice, a response blocked by the TRPV1 antagonist SB366791. H 2 O 2 -induced vasodilation was significantly inhibited in db/db and V1KO mice. H 2 O 2 caused robust SB366791-sensitive dilation in WT coronary microvessels; however, this response was attenuated in vessels from db/db and V1KO mice, suggesting H 2 O 2 -induced vasodilation occurs, in part, via TRPV1. Acute H 2 O 2 exposure potentiated capsaicin-induced CBF responses and capsaicin-mediated vasodilation in WT mice, whereas prolonged luminal H 2 O 2 exposure blunted capsaicin-induced vasodilation. Electrophysiology studies re-confirms acute H 2 O 2 exposure activated TRPV1 in HEK293A and bovine aortic endothelial cells while establishing that H 2 O 2 potentiate capsaicin-activated TRPV1 currents, whereas prolonged H 2 O 2 exposure attenuated TRPV1 currents. Verification of H 2 O 2 -mediated activation of intrinsic TRPV1 specific currents were found in isolated mouse coronary endothelial cells from WT mice and decreased in endothelial cells from V1KO mice. These data suggest prolonged H 2 O 2 exposure impairs TRPV1-dependent coronary vascular signaling. This may contribute to microvascular dysfunction and tissue perfusion deficits characteristic of diabetes.

A molecular switch for eNOS The enzyme eNOS (endothelial nitric oxide synthase) is vital for regulating vascular function as it can produce both the vasodilator nitric oxide and the vasoconstrictor superoxide. Jay Zweier and colleagues show that a modification associated with oxidant stress, S-glutathionylation, switches the enzyme from forming nitric oxide to forming superoxide. In hypertensive vessels, S-glutathionylation of eNOS is increased, and this is associated with impaired endothelium-dependent vasodilation. Oxidant stress occurs in many diseases including heart attack, stroke, diabetes and cancer. This work suggests that agents that reset this redox switch, thereby restoring normal nitric oxide synthase function, may have therapeutic potential. The enzyme eNOS is crucial for regulating vascular function as it can produce both the vasodilator nitric oxide and the vasoconstrictor superoxide. Here it is shown that a modification associated with oxidant stress, S-glutathionylation, switches the enzyme from forming nitric oxide to forming superoxide. In hypertensive vessels, S-glutathionylation of eNOS is increased and this is associated with impaired endothelium-dependent vasodilation. Endothelial nitric oxide synthase (eNOS) is critical in the regulation of vascular function, and can generate both nitric oxide (NO) and superoxide (O 2 • − ), which are key mediators of cellular signalling. In the presence of Ca 2+ /calmodulin, eNOS produces NO, endothelial-derived relaxing factor, from l -arginine ( l -Arg) by means of electron transfer from NADPH through a flavin containing reductase domain to oxygen bound at the haem of an oxygenase domain, which also contains binding sites for tetrahydrobiopterin (BH 4 ) and l -Arg 1 , 2 , 3 . In the absence of BH 4 , NO synthesis is abrogated and instead O 2 • − is generated 4 , 5 , 6 , 7 . While NOS dysfunction occurs in diseases with redox stress, BH 4 repletion only partly restores NOS activity and NOS-dependent vasodilation 7 . This suggests that there is an as yet unidentified redox-regulated mechanism controlling NOS function. Protein thiols can undergo S-glutathionylation, a reversible protein modification involved in cellular signalling and adaptation 8 , 9 . Under oxidative stress, S-glutathionylation occurs through thiol–disulphide exchange with oxidized glutathione or reaction of oxidant-induced protein thiyl radicals with reduced glutathione 10 , 11 . Cysteine residues are critical for the maintenance of eNOS function 12 , 13 ; we therefore speculated that oxidative stress could alter eNOS activity through S-glutathionylation. Here we show that S-glutathionylation of eNOS reversibly decreases NOS activity with an increase in O 2 • − generation primarily from the reductase, in which two highly conserved cysteine residues are identified as sites of S-glutathionylation and found to be critical for redox-regulation of eNOS function. We show that eNOS S-glutathionylation in endothelial cells, with loss of NO and gain of O 2 • − generation, is associated with impaired endothelium-dependent vasodilation. In hypertensive vessels, eNOS S-glutathionylation is increased with impaired endothelium-dependent vasodilation that is restored by thiol-specific reducing agents, which reverse this S-glutathionylation. Thus, S-glutathionylation of eNOS is a pivotal switch providing redox regulation of cellular signalling, endothelial function and vascular tone.

Coronary microvascular dysfunction is prevalent among people with diabetes and is correlated with cardiac mortality. Compromised endothelial-dependent dilation (EDD) is an early event in the progression of diabetes, but its mechanisms remain incompletely understood. Nitric oxide (NO) is the major endothelium-dependent vasodilatory metabolite in the healthy coronary circulation, but this switches to hydrogen peroxide (H2O2) in coronary artery disease (CAD) patients. Because diabetes is a significant risk factor for CAD, we hypothesized that a similar NO-to-H2O2 switch would occur in diabetes. Vasodilation was measured ex vivo in isolated coronary arteries from wild type (WT) and microRNA-21 (miR-21) null mice on a chow or high-fat/high-sugar diet, and B6.BKS(D)-Leprdb/J (db/db) mice using myography. Myocardial blood flow (MBF), blood pressure, and heart rate were measured in vivo using contrast echocardiography and a solid-state pressure sensor catheter. RNA from coronary arteries, endothelial cells, and cardiac tissues was analyzed via quantitative real-time PCR for gene expression, and cardiac protein expression was assessed via western blot analyses. Superoxide was detected via electron paramagnetic resonance. (1) Ex vivo coronary EDD and in vivo MBF were impaired in diabetic mice. (2) Nω-Nitro-L-arginine methyl ester, an NO synthase inhibitor (L-NAME), inhibited ex vivo coronary EDD and in vivo MBF in WT. In contrast, polyethylene glycol-catalase, an H2O2 scavenger (Peg-Cat), inhibited diabetic mouse EDD ex vivo and MBF in vivo. (3) miR-21 was upregulated in diabetic mouse endothelial cells, and the deficiency of miR-21 prevented the NO-to-H2O2 switch and ameliorated diabetic mouse vasodilation impairments. (4) Diabetic mice displayed increased serum NO and H2O2, upregulated mRNA expression of Sod1, Sod2, iNos, and Cav1, and downregulated Pgc-1α in coronary arteries, but the deficiency of miR-21 reversed these changes. (5) miR-21-deficient mice exhibited increased cardiac PGC-1α, PPARα and eNOS protein and reduced endothelial superoxide. (6) Inhibition of PGC-1α changed the mRNA expression of genes regulated by miR-21, and overexpression of PGC-1α decreased the expression of miR-21 in high (25.5 mM) glucose treated coronary endothelial cells. Diabetic mice exhibit a NO-to-H2O2 switch in the mediator of coronary EDD, which contributes to microvascular dysfunction and is mediated by miR-21. This study represents the first mouse model recapitulating the NO-to-H2O2 switch seen in CAD patients in diabetes.

Endothelial dysfunction in diabetes is generally attributed to oxidative stress, but this view is challenged by observations showing antioxidants do not eliminate diabetic vasculopathy. As an alternative to oxidative stress-induced dysfunction, we interrogated if impaired mitochondrial function in endothelial cells is central to endothelial dysfunction in the metabolic syndrome. We observed reduced coronary arteriolar vasodilation to the endothelium-dependent dilator, acetylcholine (Ach), in Zucker Obese Fatty rats (ZOF, 34 ± 15% [mean ± standard deviation] 10–3 M) compared to Zucker Lean rats (ZLN, 98 ± 11%). This reduction in dilation occurred concomitantly with mitochondrial DNA (mtDNA) strand lesions and reduced mitochondrial complex activities in the endothelium of ZOF versus ZLN. To demonstrate endothelial dysfunction is linked to impaired mitochondrial function, administration of a cell-permeable, mitochondria-directed endonuclease (mt-tat-EndoIII), to repair oxidatively modified DNA in ZOF, restored mitochondrial function and vasodilation to Ach (94 ± 13%). Conversely, administration of a cell-permeable, mitochondria-directed exonuclease (mt-tat-ExoIII) produced mtDNA strand breaks in ZLN, reduced mitochondrial complex activities and vasodilation to Ach in ZLN (42 ± 16%). To demonstrate that mitochondrial function is central to endothelium-dependent vasodilation, we introduced (via electroporation) liver mitochondria (from ZLN) into the endothelium of a mesenteric vessel from ZOF and restored endothelium-dependent dilation to vasoactive intestinal peptide (VIP at 10–5 M, 4 ± 3% vasodilation before mitochondrial transfer and 48 ± 36% after transfer). Finally, to demonstrate mitochondrial function is key to endothelium-dependent dilation, we administered oligomycin (mitochondrial ATP synthase inhibitor) and observed a reduction in endothelium-dependent dilation. We conclude that mitochondrial function is critical for endothelium-dependent vasodilation.

Mitochondrial dysfunction in obesity and diabetes can be caused by excessive production of free radicals, which can damage mitochondrial DNA. Because mitochondrial DNA plays a key role in the production of ATP necessary for cardiac work, we hypothesized that mitochondrial dysfunction, induced by mitochondrial DNA damage, uncouples coronary blood flow from cardiac work. Myocardial blood flow (contrast echocardiography) was measured in Zucker lean (ZLN) and obese fatty (ZOF) rats during increased cardiac metabolism (product of heart rate and arterial pressure, i.v. norepinephrine). In ZLN increased metabolism augmented coronary blood flow, but in ZOF metabolic hyperemia was attenuated. Mitochondrial respiration was impaired and ROS production was greater in ZOF than ZLN. These were associated with mitochondrial DNA (mtDNA) damage in ZOF. To determine if coronary metabolic dilation, the hyperemic response induced by heightened cardiac metabolism, is linked to mitochondrial function we introduced recombinant proteins (intravenously or intraperitoneally) in ZLN and ZOF to fragment or repair mtDNA, respectively. Repair of mtDNA damage restored mitochondrial function and metabolic dilation, and reduced ROS production in ZOF; whereas induction of mtDNA damage in ZLN reduced mitochondrial function, increased ROS production, and attenuated metabolic dilation. Adequate metabolic dilation was also associated with the extracellular release of ADP, ATP, and H 2 O 2 by cardiac myocytes; whereas myocytes from rats with impaired dilation released only H 2 O 2 . In conclusion, our results suggest that mitochondrial function plays a seminal role in connecting myocardial blood flow to metabolism, and integrity of mtDNA is central to this process.

Diazoxide, a mitochondrial ATP-sensitive potassium (mitoKATP) channel opener, protects the heart from ischemia-reperfusion injury. Diazoxide also inhibits mitochondrial complex II-dependent respiration in addition to its preconditioning effect. However, there are no prior studies of the role of diazoxide on post-ischemic myocardial oxygenation. In the current study, we determined the effect of diazoxide on the suppression of post-ischemic myocardial tissue hyperoxygenation in vivo, superoxide (O₂ ⁻•) generation in isolated mitochondria, and impairment of the interaction between complex II and complex III in purified mitochondrial proteins. It was observed that diazoxide totally suppressed the post-ischemic myocardial hyperoxygenation. With succinate but not glutamate/malate as the substrate, diazoxide significantly increased ubisemiquinone-dependent O₂ ⁻• generation, which was not blocked by 5-HD and glibenclamide. Using a model system, the super complex of succinate-cytochrome c reductase (SCR) hosting complex II and complex III, we also observed that diazoxide impaired complex II and its interaction with complex III with no effect on complex III. UV-visible spectral analysis revealed that diazoxide decreased succinate-mediated ferricytochrome b reduction in SCR. In conclusion, our results demonstrated that diazoxide suppressed the in vivo post-ischemic myocardial hyperoxygenation through opening the mitoKATP channel and ubisemiquinone-dependent O₂ ⁻• generation via inhibiting mitochondrial complex II-dependent respiration.

Protein thiols with regulatory functions play a critical role in maintaining the homeostasis of the redox state in mitochondria. One major host of regulatory cysteines in mitochondria is Complex I, with the thiols primarily located on its 51 kDa FMN-binding subunit. In response to oxidative stress, these thiols are expected to form intramolecular disulfide bridges as one of their oxidative post-translational modifications. Here, to test this hypothesis and gain insights into the molecular pattern of disulfide in Complex I, the isolated bovine Complex I was prepared. Superoxide (O 2 ·−) is generated by Complex I under the conditions of enzyme turnover. O 2 ·−-induced intramolecular disulfide formation at the 51, kDa subunit was determined by tandem mass spectrometry and database searching, with the latter accomplished by adaptation of the in-house developed database search engine, MassMatrix [Xu, H., et al., J. Proteome Res. 2008, 7, 138–144]. LC/MS/MS analysis of tryptic/chymotryptic digests of the 51 kDa subunit from alkylated Complex I revealed that four specific cysteines (C 125, C 142, C 187, and C 206) of the 51 kDa subunit were involved in the formation of mixed intramolecular disulfide linkages. In all, three cysteine pairs were observed: C 125/C 142, C 187/C 206, and C 142/C 206. The formation of disulfide bond was subsequently inhibited by superoxide dismutase, indicating the involvement of O 2 ·−. These results elucidated by mass spectrometry indicate that the residues of C 125, C 142, C 187, and C 206 are the specific regulatory cysteines of Complex I and they participate in the oxidative modification with disulfide formation under the physiological or pathophysiological conditions of oxidative stress. Mass spectrometry coupled with the MassMatrix program was used to probe the molecular pattern of oxidant-induced disulfide linkage in the FMN-binding subunit of mitochondrial Complex I.