Uncoupled Oxidation Research Articles

POSTTRANSLATIONAL MODIFICATIONS OF THE SULFHYDRYL GROUP OF THE CYSTEINE RESIDUE OF GLYCERALDEHYDE-3-PHOSPHATE DEHYDROGENASE

This review considers the main types of oxidative posttranslational modi cations of the glycolytic enzyme glyceraldehyde-3-phosphate dehydrogenase (GAPDH) targeting the sulfhydryl group of the catalytic cysteine residue Cys152. The highly reactive sulfhydryl group of Cys152 in the active centre of GAPDH undergoes oxidation and S-nitrosylation, leading to enzyme inactivation and destabilization. Upon reversible oxidation of the sulfhydryl group to form cysteine-sulfenic acid, the enzyme loses dehydrogenase activity, but gains the ability to catalyze the acyl-phosphatase reaction. Hydrolysis of the product of the dehydrogenase reaction, 1,3-diphosphoglycerate, under the action of the oxidized GAPDH leads to uncoupling of oxidation and phosphorylation at this stage of glycolysis. The action of nitric oxide results in S-nitrosylation of Cys152 GAPDH and the subsequent formation of cysteine-sulfenic acid due to hydrolysis of the S-NO-group. Data are presented on the relationship between S-nitrosylation of the catalytic Cys152 of GAPDH and its oxidation followed by S-glutathionylation of the enzyme at Cys152. The role of posttranslational modi cations of the sulfhydryl group of the catalytic cysteine residue in the regulation of enzyme activity, as well as the mechanisms ensuring the reversibility of such modi cations are discussed.

Association of a high rate of glycolysis and the activity of the uncoupling of oxidation and phosphorylation in the blood cells of patients with late-stage knee osteoarthritis and the development of postoperative pain

Objective: to investigate the relationship between the expression of genes mediating cellular energy production and the development of chronic postoperative pain (CPP) after total knee arthroplasty (TKA) in patients with osteoarthritis (OA).Material and methods. Prior to TKA, the blood of 50 patients with stage III–IV knee OA and complaints of constant pain and joint dysfunction was analyzed. The control group consisted of 26 healthy individuals. Pain intensity was assessed using a visual analogue scale (VAS), a short BPI questionnaire and the WOMAC index, and the presence of neuropathic pain was assessed using the DN4 and PainDETECT questionnaires. The development of CPP was determined 3 and 6 months after TKA. Total RNA isolated from blood was used to determine the expression of PKM2, LDH, SDH, AMPKα, PDH, IDH, MDH and ATP synthase genes by real-time quantitative reverse transcriptase-polymerase chain reaction.Results and discussion. CPP ≥30 mm according to VAS was detected in 17 patients. Before TKA, the expression of all analyzed genes was significantly increased compared to that of the control group. However, there were no differences in clinical, pain-related and functional indicators in the analyzed group of patients with OA. Before surgery, patients who subsequently developed CPP had significantly higher expression of genes related to glycolysis (PKM2, LDH), Krebs cycle – KC (SDH) and master regulator of energy metabolism (AMPKα) than patients who were satisfied with the results of TKA. At the same time, no differences were found in the expression of PDH and other KC enzyme genes (IDH, MDH) and ATP synthase in patients with and without CPP.Conclusion. The development of CPP is associated with a higher rate of glycolysis and energy deficiency, presumably due to the higher uncoupling activity of oxidation and phosphorylation that can be observed before TKA.

Alterations of hepatic energy metabolism in murine models of obesity, diabetes and fatty liver diseases.

Disturbed hepatic energy metabolism contributes to non-alcoholic fatty liver (NAFLD), but the development of changes over time and obesity- or diabetes-related mechanisms remained unclear. Two-day old male C57BL/6j mice received streptozotocin (STZ) or placebo (PLC) and then high-fat (HFD) or regular chow diet (RCD) from week 4 (W4) to either W8 or W16, yielding control [CTRL=PLC+RCD], diabetes [DIAB=STZ+RCD], obesity [OBES=PLC+HFD] and diabetes-related non-alcoholic steatohepatitis [NASH=STZ+HFD] models. Mitochondrial respiration was measured by high-resolution respirometry and insulin-sensitive glucose metabolism by hyperinsulinemic-euglycemic clamps with stable isotope dilution. NASH showed higher steatosis and NAFLD activity already at W8 and liver fibrosis at W16 (all p<0.01 vs CTRL). Ballooning was increased in DIAB and NASH at W16 (p<0.01 vs CTRL). At W16, insulin sensitivity was 47%, 58% and 75% lower in DIAB, NASH and OBES (p<0.001 vs CTRL). Hepatic uncoupled fatty acid oxidation (FAO)-associated respiration was reduced in OBES at W8, but doubled in DIAB and NASH at W16 (p<0.01 vs CTRL) and correlated with biomarkers of unfolded protein response (UPR), oxidative stress and hepatic expression of certain enzymes (acetyl-CoA carboxylase 2, Acc2; carnitine palmitoyltransferase I, Cpt1a). Tricarboxylic acid cycle (TCA)-driven respiration was lower in OBES at W8 and doubled in DIAB at W16 (p<0.0001 vs CTRL), which positively correlated with expression of genes related to lipolysis. Hepatic mitochondria adapt to various metabolic challenges with increasing FAO-driven respiration, which is linked to dysfunctional UPR, systemic oxidative stress, insulin resistance and altered lipid metabolism. In a diabetes model, higher TCA-linked respiration reflected mitochondrial adaptation to greater hepatic lipid turnover. Funding bodies that contributed to this study were listed in the acknowledgements section.

The ketogenic diet promotes triacylglycerol recycling in white adipose tissue and uncoupled fat oxidation in brown adipose tissue, but does not reduce adiposity in rats

The purpose of this study was to determine whether the weight-reducing and fat burning effects of the ketogenic diet (KD) could be attributed to alterations in the energy dissipating pathways of brown adipose tissue (BAT) uncoupled oxidation, and white adipose tissue (WAT) browning and triacylglycerol (TAG) recycling. To investigate this, male Wistar rats were fed one of the following three diets for either 8 or 16 weeks: a standard chow (SC), a high-fat, sucrose-enriched (HFS) obesogenic diet, or a KD. At the end of the intervention, subcutaneous inguinal (Sc Ing) and epididymal (Epid) fat, and interscapular and aortic BAT (iBAT and aBAT, respectively) were extracted. These tissues were used for the analysis of proteins involved in WAT browning and thermogenesis. Isolated adipocytes from WAT were assayed for basal and isoproterenol (Iso)-stimulated lipolysis and basal and insulin-stimulated lipogenesis, and BAT adipocytes were assayed for the determination of coupled and uncoupled glucose and palmitate oxidation. Adiposity similarly increased in HFS- and KD-fed rats at weeks 8 and 16. However, in HFS-fed animals insulin-stimulated lipogenesis and Iso-stimulated lipolysis were impaired in WAT adipocytes, whereas in KD-fed animals these pathways remained intact. The KD also significantly elevated WAT glycerol kinase levels, and favored TAG recycling under conditions of enhanced lipolysis. In BAT, the KD significantly increased uncoupling protein-1 levels and uncoupled fat oxidation. In summary, the KD preserved insulin sensitivity and lipolytic capacity in WAT and also upregulated energy-dissipating pathways in BAT, but it was not sufficient to prevent an increase in adiposity.

Photoinhibition of comammox reaction in Nitrospira inopinata in a dose- and wavelength-dependent manner.

Apparent contribution of complete ammonia-oxidizing organisms (comammox) to the global nitrogen cycle highlights the necessity for understanding niche differentiation of comammox bacteria among other ammonia oxidizers. While the high affinity for ammonia of the comammox species Nitrospira inopinata suggests their niche partitioning is expected to be centered in oligotrophic environments, their absence in nutrient-depleted environments (such as the oceans) suggests that other (abiotic) factors might control their distribution and spatial localization within microbial communities. Many ammonia- and nitrite-oxidizing organisms are sensitive to light; however, the photosensitivity of comammox has not been explored. Since comammox bacteria encode enzymatic machinery homologous to canonical ammonia-and nitrite-oxidizers, we hypothesized that comammox N. inopinata, the only available pure culture of this group of microorganisms, may be inhibited by illumination in a similar manner. We evaluated the impact of light intensity, wavelength, and duration on the degree of photoinhibition for cultures of the comammox species N. inopinata and the soil ammonia-oxidizing archaea Nitrososphaera viennensis. Both species were highly sensitive to light. Interestingly, mimicking diurnal light exposure caused an uncoupling of ammonia and nitrite oxidation in N. inopinata, indicating nitrite oxidation might be more sensitive to light exposure than ammonia oxidation. It is likely that light influences comammox spatial distribution in natural environments such as surface fresh waters according to diurnal cycles, light attenuation coefficients, and the light penetration depths. Our findings therefore provide ecophysiological insights for further studies on comammox both in field and laboratory settings.

An obesogenic diet impairs uncoupled substrate oxidation and promotes whitening of the brown adipose tissue in rats.

Brown adipose tissue (BAT) is richin mitochondria containing uncoupling protein 1 (UCP1), and dissipates energy through thermogenesis. However, even though BAT mass and its UCP1 content increase in rodents chronically fed a high-fat sucrose-enriched (HFS) diet, marked expansion of adiposity still occurs in these animals, suggesting insufficient BAT-mediated HFS diet-induced thermogenesis. Thus, the objective of this study was to investigate the metabolic and molecular mechanisms that regulate BAT thermogenesis in HFS-induced obesity. To accomplish this, rats were fed either a standard chow or HFS diet for 8weeks. Subsequently, glucose and fatty acidmetabolism and the molecular mechanisms underlying these processes were assessed in freshly isolated primary BAT adipocytes. Despite increasing BAT mass and its UCP1 content, the HFS diet reduced uncoupled glucose and palmitate oxidation in BAT adipocytes. It also markedly diminished tyrosine hydroxylase content and lipolysis in these cells. Conversely, glucose uptake, lactate production, glycerol incorporation into lipids, palmitate incorporation into triacylglycerol (TAG), phosphoenolpyruvate carboxykinase and glycerol kinase levels, and lipoprotein lipase and cluster of differentiation 36 gene expression were increased. In summary, a HFS diet enhanced glyceroneogenesis and shifted BAT metabolism toward TAG synthesis by impairing UCP1-mediated substrate oxidation and by enhancing fatty acid esterification inintact brown adipocytes. These adaptive metabolic responses to chronic HFS feeding attenuated BAT thermogenic capacity and favoured the development of obesity. KEY POINTS: Despite increasing brown adipose tissue (BAT) mass and levels of thermogenic proteins such as peroxisome proliferator-activated receptor γ coactivator 1α, carnitine palmitoyltransferase 1B and uncoupling protein 1 (UCP1), an obesogenic high-fat sucrose-enriched (HFS) diet attenuated uncoupled glucose and fatty acid oxidation in brown adipocytes. Brown adipocytes diverted glycerol and fatty acids toward triacylglycerol (TAG) synthesis by elevating the cellular machinery that promotes fatty acid uptake along with phosphoenolpyruvate carboxykinase and glycerol kinase levels. The HFS diet increased glucose uptake that supported lactate production and provided substrate for glyceroneogenesis and TAG synthesis in brown adipocytes. Impaired UCP-1-mediated thermogenic capacity and enhanced TAG storage in BAT adipocytes were consistent with reduced adipose triglyceride lipase and tyrosine hydroxylase levels in HFS diet-fed animals.

DISTURBANCE OF PRO-OXIDANT-ANTIOXIDANT BALANCE IN RATS WITH CEREBRAL ISCHEMIA

Excess of reactive oxygen can lead to membrane damage, accumulation of lipid, protein, and nucleic acid oxidation products, deficiency of reduced pyridine nucleotides and phospholipids of mitochondrial membranes, and then to electrolyte imbalance, mitochondrial swelling, uncoupling of oxidation and phosphorylation processes, and ischemic neuronal death. Thus, the study of oxidative stress and antioxidant system activity is relevant.&#x0D; The aim of the study is to examine the changes in the pro-oxidant-antioxidant balance in rats with ischemic brain damage of different degrees of severity (subtotal and total cerebral ischemia).&#x0D; Materials and Methods. The experiments were performed on 30 male outbred white rats weighing 260±20 g in compliance with the requirements of the Directive of the European Parliament and the Council of the European Union No. 2010/63/EU of September 22, 2010 on the protection of animals used for scientific purposes.&#x0D; Results. A more significant decrease in the content of total SH-groups of proteins and glutathione (by 58 (51; 64) % (p&lt;0.05)), and GSH concentration (by 29 (19; 35) % (p&lt;0.05)) was observed under 24-hour subtotal brain ischemia (SBI) compared with 1-hour SBI. Changes in the glutathione peroxidase activity were multidirectional: in 1-hour SBI, the activity increased by 12 (9; 18) % (p&lt;0.05compared to the control level, and in 24-hour SBI, it decreased by 74 (67; 81) % (p&lt;0.05). In 1-hour SBI, the content of total SH-groups of proteins and glutathione was higher by 60 (54; 65) % (p&lt;0.05), and GSH concentration was higher by 42 (39; 56) % (p&lt;0.05) compared with 1-hour total brain ischemia (TBI). The content of products that react with thiobarbituric acid increased by 59 (51; 63) % (p&lt;0.05). In 24-hout SBI, the content of total SH-groups of proteins and glutathione was higher by 36 (29; 45) % (p&lt;0.05), and GSH concentration was higher by 63 (59; 75) % (p&lt;0.05) compared with 24-hour TBI. The content of products that react with thiobarbituric acid increased by 83 (78; 91) % (p&lt;0.05). The glutathione peroxidase activity in TBI was equal to zero.&#x0D; Conclusions. Thus, the most pronounced disturbances in the pro-oxidant-antioxidant balance were observed in 24-hour TBI. Similar, but less pronounced disturbances were observed in 24-hour SBI.

Mitochondrial Adaptation to Diet and Swimming Activity in Gilthead Seabream: Improved Nutritional Efficiency.

Sustained exercise promotes growth in different fish species, and in gilthead seabream we have demonstrated that it improves nutrient use efficiency. This study assesses for differences in growth rate, tissue composition and energy metabolism in gilthead seabream juveniles fed two diets: high-protein (HP; 54% protein, 15% lipid) or high energy (HE; 50% protein, 20% lipid), under voluntary swimming (VS) or moderate-to-low-intensity sustained swimming (SS) for 6 weeks. HE fed fish under VS conditions showed lower body weight and higher muscle lipid content than HP fed fish, but no differences between the two groups were observed under SS conditions. Irrespective of the swimming regime, the white muscle stable isotopes profile of the HE group revealed increased nitrogen and carbon turnovers. Nitrogen fractionation increased in the HP fed fish under SS, indicating enhanced dietary protein oxidation. Hepatic gene expression markers of energy metabolism and mitochondrial biogenesis showed clear differences between the two diets under VS: a significant shift in the COX/CS ratio, modifications in UCPs, and downregulation of PGC1a in the HE-fed fish. Swimming induced mitochondrial remodeling through upregulation of fusion and fission markers, and removing almost all the differences observed under VS. In the HE-fed fish, white skeletal muscle benefited from the increased energy demand, amending the oxidative uncoupling produced under the VS condition by an excess of lipids and the pro-fission state observed in mitochondria. Contrarily, red muscle revealed more tolerant to the energy content of the HE diet, even under VS conditions, with higher expression of oxidative enzymes (COX and CS) without any sign of mitochondrial stress or mitochondrial biogenesis induction. Furthermore, this tissue had enough plasticity to shift its metabolism under higher energy demand (SS), again equalizing the differences observed between diets under VS condition. Globally, the balance between dietary nutrients affects mitochondrial regulation due to their use as energy fuels, but exercise corrects imbalances allowing practical diets with lower protein and higher lipid content without detrimental effects.

Induction of UCP1 and thermogenesis by a small molecule via AKAP1/PKA modulation

Strategies to increase energy expenditure are an attractive approach to reduce excess fat storage and body weight to improve metabolic health. In mammals, uncoupling protein-1 (UCP1) in brown and beige adipocytes uncouples fatty acid oxidation from ATP generation in mitochondria and promotes energy dissipation as heat. We set out to identify small molecules that enhance UCP1 levels and activity using a high-throughput screen of nearly 12,000 compounds in mouse brown adipocytes. We identified a family of compounds that increase Ucp1 expression and mitochondrial activity (including un-coupled respiration) in mouse brown adipocytes and human brown and white adipocytes. The mechanism of action may be through compound binding to A kinase anchoring protein (AKAP) 1, modulating its localization to mitochondria and its interaction with protein kinase A (PKA), a known node in the β-adrenergic signaling pathway. In mice, the hit compound increased body temperature, UCP1 protein levels, and thermogenic gene expression. Some of the compound effects on mitochondrial function were UCP1- or AKAP1-independent, suggesting compound effects on multiple nodes of energy regulation. Overall, our results highlight a role for AKAP1 in thermogenesis, uncoupled respiration, and regulation energy balance.

Abstract MP124: De-palmitoylation of Cysteine 202 of Cyclophilin-D by Calcium is Important for the Activation of the Permeability Transition Pore

Cyclophilin-D (CypD) is a well-known regulator of the mitochondrial permeability transition pore (PTP), the main effector of cardiac ischemia/reperfusion (I/R) injury characterized by oxidative stress and calcium overload. However, the mechanism by which CypD activates PTP is poorly understood. Cysteine 202 of CypD (C202) is highly conserved across species and can undergo redox-sensitive post-translational modifications, such as S-nitrosylation and oxidation. To study the importance of C202, we developed a knock-in mouse model using CRISPR where CypD-C202 was mutated to a serine (C202S). Hearts from these mice are protected against I/R injury. We found C202 to be abundantly S-palmitoylated under baseline conditions while C202 was de-palmitoylated during ischemia in WT hearts. To further investigate the mechanism of de-palmitoylation during ischemia, we considered the increase of matrix calcium, oxidative stress and uncoupling of ATP synthesis from the electron transport chain. We tested the effects of these conditions on the palmitoylation of CypD in isolated cardiac mitochondria. The palmitoylation of CypD was assessed using a resin-assisted capture (Acyl-RAC). We report that oxidative stress (phenylarsenide) and uncoupling (CCCP) had no effect on CypD palmitoylation (p&gt;0.05, n=3 and n=7 respectively). However, calcium overload led to de-palmitoylation of CypD to the level observed at the end ischemia (1±0.10 vs 0.63±0.09, p=0.012, n=9). To further test the hypothesis that calcium regulates S-palmitoylation of CypD we measured S-palmitoylation of CypD in non-perfused heart lysates from global germline mitochondrial calcium uniporter knock-out mice (MCU-KO), which have reduced mitochondrial calcium and we found an increase in S-palmitoylation of CypD (WT 1±0.04 vs MCU-KO 1.603±0.11, p&lt;0.001, n=6). The data are consistent with the hypothesis that C202 is important for the CypD mediated activation of PTP. Ischemia leads to increased matrix calcium which in turn promotes the de-palmitoylation of CypD on C202. The now free C202 can further be oxidized during reperfusion leading to the activation of PTP. Thus, S-palmitoylation and oxidation of CypD-C202 possibly target CypD to the PTP, making them potent regulators of cardiac I/R injury.

Changes in Myocardial Metabolism Preceding Sudden Cardiac Death.

Heart disease is widely recognized as a major cause of death worldwide and is the leading cause of mortality in the United States. Centuries of research have focused on defining mechanistic alterations that drive cardiac pathogenesis, yet sudden cardiac death (SCD) remains a common unpredictable event that claims lives in every age group. The heart supplies blood to all tissues while maintaining a constant electrical and hormonal feedback communication with other parts of the body. As such, recent research has focused on understanding how myocardial electrical and structural properties are altered by cardiac metabolism and the various signaling pathways associated with it. The importance of cardiac metabolism in maintaining myocardial function, or lack thereof, is exemplified by shifts in cardiac substrate preference during normal development and various pathological conditions. For instance, a shift from fatty acid (FA) oxidation to oxygen-sparing glycolytic energy production has been reported in many types of cardiac pathologies. Compounded by an uncoupling of glycolysis and glucose oxidation this leads to accumulation of undesirable levels of intermediate metabolites. The resulting accumulation of intermediary metabolites impacts cardiac mitochondrial function and dysregulates metabolic pathways through several mechanisms, which will be reviewed here. Importantly, reversal of metabolic maladaptation has been shown to elicit positive therapeutic effects, limiting cardiac remodeling and at least partially restoring contractile efficiency. Therein, the underlying metabolic adaptations in an array of pathological conditions as well as recently discovered downstream effects of various substrate utilization provide guidance for future therapeutic targeting. Here, we will review recent data on alterations in substrate utilization in the healthy and diseased heart, metabolic pathways governing cardiac pathogenesis, mitochondrial function in the diseased myocardium, and potential metabolism-based therapeutic interventions in disease.

Temperature decouples ammonia and nitrite oxidation in greenhouse vegetable soils

The influence of temperature on soil ammonia (NH3) and nitrite (NO2−) oxidation and related NO2− accumulation in soils remain unclear. The soil potential NH3 oxidation (PAO) and NO2− oxidation (PNO) rates were evaluated over a temperature gradient of 5–45 °C in six greenhouse vegetable soils using inhibitors. The values of temperature sensitivity traits such as temperature minimum (Tmin), temperature optimum (Topt), and maximum absolute temperature sensitivity (Tm_sens) were also fitted to the square root growth (SQRT) and macromolecular rate theory (MMRT) models. The ammonia-oxidizing archaea (AOA) and bacteria (AOB) were determined by quantifying amoA, and nitrite-oxidizing bacteria (NOB) were determined by quantifying the nxrA and nxrB. Both models identified that Topt for PAO (34.0 °C) was significantly greater than that for PNO (26.0 °C). The Tm_sens (23.4 ± 2.1 °C) and Tmin (1.0 ± 2.0 °C) for PAO were higher than those for PNO (16.8 ± 3.2 °C and − 11.7 ± 6.7 °C). PAO was positively correlated with AOB-amoA at 20–30 °C and with AOA-amoA at 30–35 °C, while PNO was positively correlated with nxrB at 5–30 °C. Additionally, NO2− and N2O were positively correlated with the (AOA + AOB amoA) to (nxrA + nxrB) ratio, and the concentration of N2O was positively correlated with NO2− accumulation. These results highlight that elevated temperatures resulted in the uncoupling of NH3 oxidation and NO2− oxidation, leading to NO2− accumulation, which could stimulate N2O emissions.

Effects of psychoactive drugs on cellular bioenergetic pathways

Objectives To investigate the actions of lithium, valproate, lamotrigine and quetiapine on bioenergetic pathways in cultured NT2-N neuronal-like cells and C8-B4 microglial cells. Methods NT2-N and C8-B4 cells were cultured and treated with lithium (2.5 mM), valproate (0.5 mM), quetiapine (0.05 mM) or lamotrigine (0.05 mM) for 24 hours. Gene expression and the mitochondrial bioenergetic profile were measured in both cell lines. Results In NT2-N cells, valproate increased oxidative phosphorylation (OXPHOS) gene expression, mitochondrial uncoupling and maximal respiratory capacity, while quetiapine decreased OXPHOS gene expression and respiration linked to ATP turnover, as well as decreasing the expression of genes in the citric acid cycle. Lamotrigine decreased OXPHOS gene expression but had no effect on respiration, while lithium reduced the expression of genes in the citric acid cycle. In C8-B4 cells, valproate and lithium increased OXPHOS gene expression, and valproate increased basal respiratory rate and maximal and spare respiratory capacities. In contrast, quetiapine significantly reduced basal respiratory rate and maximal and spare respiratory capacities. Conclusions Overall our data suggest that some drugs used to treat neuropsychiatric and affective disorders have actions on a range of cellular bioenergetic processes, which could impact their effects in patients.

Brown Adipose Tissue, Thermogenesis, and Obesity: A Review of Literature

The white adipose tissue (WAT) mass in adult humans ranges between 10-35 kg. The cells are normally sensitive to insulin in the fed state, and to glucagon and adrenaline in fasting or during exercise. Well-fed sedentary individuals are prone to weight gain as they fall victims to the anabolic mechanisms led mainly by insulin. Brown adipose tissue (BAT), by contrast, stores smaller amounts of triglycerides in multi-locular droplets, is highly vascularized and its cells are rich in unique mitochondria which are capable of uncoupling oxidation from phosphorylation or ATP formation. The tissue is innerved by the sympathetic nervous system and is highly sensitive to iodothyronines. It releases heat in the body in response to sympathetic activity. BAT unique mitochondria express numerous cristae and, unlike ordinary mitochondria on other body cells including WAT cells, they express uncoupling protein-1 (UCP-1, or thermogenin). UCP-1 allows the mitochondria to oxidize more fat and glucose as they escape the controlling mechanisms that depend on the coupling of oxidation to the demand for ATP, i.e., uncouples oxidative phosphorylation. Thermogenin (UCP-1) is a natural un-coupler of oxidative phosphorylation as it dissipates the proton gradient generated across the inner mitochondrial membrane, which is required to attain a certain level for the activation of ATP synthesis in mitochondrial matrix. In BAT, ATP synthesis is inhibited, as the protons are dissipated, and most energy is released as heat. The different proportions and activity of BAT and WAT in different individuals might explain why some people are more prone to weight gain, and find it difficult to lose weight, than others; and also explains the tendency for weight gain as individuals get older. New approaches in the battle against obesity, metabolic syndrome and type 2 diabetes mellitus are expected through better understanding of how this balance between WAT and BAT is controlled. Interestingly, long term adrenergic stimulation of WAT induces browning of some white adipocytes, and the tissue gradually turns into &amp; quot; beige &amp; quot; adipose tissue, which shares characteristics of brown adipose tissue like thermogenesis, larger number of mitochondria and smaller lipid droplets, all developing in a gradual way. The adipose tissue interconverts its cell types in order to adapt for the changing metabolic balance and other stimuli. This phenomenon is currently incompletely understood, albeit significant for our understanding of obesity, metabolic syndrome and type 2 diabetes mellitus and many consequent complications of insulin resistance. Moreover, the nervous system is involved in the regulation of WAT and BAT through effects on fat cell proliferation, differentiation, trans-differentiation and apoptosis. The brain interacts with different adipocytes and adipokines in the pathogenesis of obesity, type 2 diabetes mellitus, anorexia, cachexia and other syndromes. This review will target many of these aspects in an attempt to draw more attention in the direction of this major health issue.

414-P: Insulin Secretion Capacity Relates to Human Ventricular Myocardial Mitochondrial Function

Hyperinsulinemia and impaired insulin secretion are potential contributors to the development of diabetes-related heart failure. Interference with ventricular myocardial mitochondria might play a key pathogenic role, but human data confirming these connections are limited. We hypothesized that ventricular myocardial mitochondrial function is impaired in hyperinsulinemia but is positively related to insulin secretion capacity. We recruited 32 heart transplant recipients without heart failure or antihyperglycemic treatment (age: 53.5±13.3years; BMI: 24.8±2.9kg/m²; HbA1c: 5.9±0.7%). We measured serum insulin levels and blood glucose every 30 minutes for three hours after an oral 75g glucose uptake. Seven patients were newly diagnosed with type 2 diabetes and six had impaired glucose tolerance. High-resolution respirometry was performed on transcatheter ventricular endomyocardial biopsies to determine mitochondrial oxidative capacity, coupling efficiency (respiratory control ratio, RCR) and intrinsic uncoupling (leak control ratio, LCR). Fasting serum insulin, a marker of insulin resistance, was inversely associated with ventricular myocardial mitochondrial RCR (r=-0.39; p=0.02) and positively with LCR (r=0.42; p=0.02), but not with oxidative capacity (p=0.34). Maximum increase of insulin in proportion to fasting levels strongly related to ventricular myocardial mitochondrial RCR (r=0.62; p=0.0001) and inversely to LCR (r=-0.46; p=0.01). This insulin secretory response related to fatty acid-dependent respiration (r=0.35; p=0.05) and tended to relate to combined oxidative capacity from fatty acid and glycolytic substrates (r=0.31; p=0.09). Maximum insulin response following glucose ingestion relates to cardiac mitochondrial efficiency and oxidative capacity, while insulin resistance might affect cardiac energy metabolism. These data suggest a pathogenic role of hyperinsulinemia as well as beta cell failure in diabetes-related heart failure. Disclosure E. Zweck: None. D. Scheiber: None. S. Albermann: None. J. Borger: None. T. Jelenik: None. D. Pesta: None. P. Horn: None. U. Boeken: None. P. AKhyari: Research Support; Self; Abbott, Edwards Lifesciences Corporation, Medtronic. M. Kelm: None. M. Roden: Advisory Panel; Self; Boehringer Ingelheim Pharmaceuticals, Inc., Poxel SA, Servier. Board Member; Self; Eli Lilly and Company. Research Support; Self; Boehringer Ingelheim Pharmaceuticals, Inc., Sanofi. Speaker's Bureau; Self; Novo Nordisk Inc. R. Westenfeld: None. J. Szendroedi: None. Funding German Research Foundation (CRC1116); Heinrich-Heine University

The effect of a physiological increase in temperature on mitochondrial fatty acid oxidation in rat myofibers.

We investigated the effect of temperature increase on mitochondrial fatty acid (FA) and carbohydrate oxidation in the slow-oxidative skeletal muscles (soleus) of rats. We measured mitochondrial respiration at 35°C and 40°C with the physiological substrates pyruvate + 4 mM malate (Pyr) and palmitoyl-CoA (PCoA) + 0.5 mM malate + 2 mM carnitine in permeabilized myofibers under nonphosphorylating () or phosphorylating () conditions. Mitochondrial efficiency was calculated by the respiratory control ratio (RCR = /). We used guanosine triphosphate (GTP), an inhibitor of uncoupling protein (UCP), to study the mechanisms responsible for alterations of mitochondrial efficiency. We measured hydrogen peroxide (H2O2) production under nonphosphorylating and phosphorylating conditions at both temperatures and substrates. We studied citrate synthase (CS) and 3-hydroxyl acyl coenzyme A dehydrogenase (3-HAD) activities at both temperatures. Elevating the temperature from 35°C to 40°C increased PCoA- and decreased PCoA-RCR, corresponding to the uncoupling of oxidative phosphorylation (OXPHOS). GTP blocked the heat-induced increase of PCoA-. Rising temperature moved toward a Pyr- increase, without significance. Heat did not alter H2O2 production, resulting from either PCoA or Pyr oxidation. Heat induced an increase in 3-HAD but not in CS activities. In conclusion, heat induced OXPHOS uncoupling for PCoA oxidation, which was at least partially mediated by UCP and independent of oxidative stress. The classically described heat-induced glucose shift may actually be mostly due to a less efficient FA oxidation. These findings raise questions concerning the consequences of heat-induced alterations in mitochondrial efficiency of FA metabolism on thermoregulation.NEW & NOTEWORTHY Ex vivo exposure of skeletal myofibers to heat uncouples substrate oxidation from ADP phosphorylation, decreasing the efficiency of mitochondria to produce ATP. This heat effect alters fatty acids (FAs) more than carbohydrate oxidation. Alteration of FA oxidation involves uncoupling proteins without inducing oxidative stress. This alteration in lipid metabolism may underlie the preferential use of carbohydrates in the heat and could decrease aerobic endurance.

Alpha Lipoic Acid Improves Endothelial Function and Oxidative Stress in Mice Exposed to Chronic Intermittent Hypoxia

Objective Obstructive sleep apnea (OSA) is characterized by recurrent airway collapse that causes chronic intermittent hypoxia (CIH). OSA is associated with systemic inflammation and oxidative stress resulting in endothelial dysfunction and cardiovascular disease (CVD). Alpha lipoic acid (ALA) is a potent antioxidant with anti-inflammatory properties. We hypothesized that dietary ALA can improve endothelial function of mice exposed to CIH. Methods Mice were exposed to either CIH or intermittent air (IA) and treated with dietary ALA (0.2% w/w) or a regular chow diet for 8 weeks. Endothelial function, endothelial nitric oxide (eNOS) uncoupling, systemic oxidative stress, systemic inflammation, aortic expression of inflammatory cytokines, and antioxidant enzymes were measured after 8 weeks. Results Mice exposed to CIH exhibited endothelial dysfunction accompanied by systemic oxidative stress and inflammation as well as increased aortic expression of inflammatory cytokines. Furthermore, CIH led to eNOS uncoupling. Treatment with dietary ALA reversed endothelial dysfunction in mice exposed to CIH, lowered systemic oxidative stress and inflammation, prevented the increases of inflammatory cytokine gene expression, increased the expression of antioxidant enzymes, and preserved eNOS in a coupled state. Conclusion ALA attenuates endothelial dysfunction by preventing oxidative stress and inflammation and restoring nitric oxide bioavailability in mice exposed to CIH. Our data suggests the potential beneficial use of ALA as adjunctive therapy in OSA.

Cold acclimation enhances UCP1 content, lipolysis, and triacylglycerol resynthesis, but not mitochondrial uncoupling and fat oxidation, in rat white adipocytes.

The objective of this study was to investigate whether cold-induced browning of the subcutaneous (Sc) inguinal (Ing) white adipose tissue (WAT) increases the capacity of this tissue to oxidize fatty acids through uncoupling protein 1 (UCP1)-mediated thermogenesis. To accomplish that, rats were acclimated to cold (4°C for 7 days). Subsequently, interscapular and aortic brown adipose tissues (iBAT and aBAT, respectively), epididymal (Epid), and Sc Ing WAT were used for adipocyte isolation. In BAT adipocytes, cold acclimation increased UCP1 content and palmitate oxidation either in the absence or presence of oligomycin, whereas in Sc Ing adipocytes glucose and palmitate oxidation were not affected, although multilocular adipocytes were formed and UCP1 content increased upon cold acclimation in the WAT. Furthermore, isoproterenol-stimulated cold Sc Ing adipocytes exhibited significantly lower rates of palmitate oxidation than control cells when exposed to oligomycin. These findings provide evidence that, despite increasing UCP1 levels, cold acclimation essentially reduced mitochondrial uncoupling-mediated fat oxidation in Sc Ing adipocytes. Conversely, glycerol kinase and phosphoenolpyruvate carboxykinase levels, isoproterenol-induced lipolysis, as well as glycerol and palmitate incorporation into lipids significantly increased in these cells. Therefore, instead of UCP1-mediated mitochondrial uncoupling, cold acclimation increased the capacity of Sc Ing adipocytes to export fatty acids and enhanced key components of the triacylglycerol resynthesis pathway in the Sc Ing WAT.

Can All Major ROS Forming Sites of the Respiratory Chain Be Activated By High FADH2 /NADH Ratios?: Ancient evolutionary constraints determine mitochondrial ROS formation.

Aspects of peroxisome evolution, uncoupling, carnitine shuttles, supercomplex formation, and missing neuronal fatty acid oxidation (FAO) are linked to reactive oxygen species (ROS) formation in respiratory chains. Oxidation of substrates with high FADH2 /NADH (F/N) ratios (e.g., FAs) initiate ROS formation in Complex I due to insufficient availability of its electron acceptor (Q) and reverse electron transport from QH2 , e.g., during FAO or glycerol-3-phosphate shuttle use. Here it is proposed that the Q-cycle of Complex III contributes to enhanced ROS formation going from low F/N ratio substrates (glucose) to high F/N substrates. This contribution is twofold: 1) Complex III uses Q as substrate, thus also competing with Complex I; 2) Complex III itself will produce more ROS under these conditions. I link this scenario to the universally observed Complex III dimerization. The Q-cycle of Complex III thus again illustrates the tension between efficient ATP generation and endogenous ROS formation. This model can explain recent findings concerning succinate and ROS-induced uncoupling.

LC-MS/MS Proteoform Profiling Exposes Cytochrome c Peroxidase Self-Oxidation in Mitochondria and Functionally Important Hole Hopping from Its Heme.

LC-MS/MS profiling reveals that the proteoforms of cytochrome c peroxidase (Ccp1) isolated from respiring yeast mitochondria are oxidized at numerous Met, Trp, and Tyr residues. In vitro oxidation of recombinant Ccp1 by H2O2 in the absence of its reducing substrate, ferrocytochrome c, gives rise to similar proteoforms, indicating uncoupling of Ccp1 oxidation and reduction in mitochondria. The oxidative modifications found in the Ccp1 proteoforms are consistent with radical transfer (hole hopping) from the heme along several chains of redox-active residues (Trp, Met, Tyr). These modifications delineate likely hole-hopping pathways to novel substrate-binding sites. Moreover, a decrease in recombinant Ccp1 oxidation by H2O2 in vitro in the presence of glutathione supports a protective role for hole hopping to this antioxidant. Isolation and characterization of extramitochondrial Ccp1 proteoforms reveals that hole hopping from the heme in these proteoforms results in selective oxidation of the proximal heme ligand (H175) and heme labilization. Previously, we demonstrated that this labilized heme is recruited for catalase maturation (Kathiresan, M.; Martins, D.; English, A. M. Respiration triggers heme transfer from cytochrome c peroxidase to catalase in yeast mitochondria. Proc. Natl. Acad. Sci. U. S. A. 2014, 111, 17468-17473; DOI: 10.1073/pnas.1409692111 ). Following heme release, apoCcp1 exits mitochondria, yielding the extramitochondrial proteoforms that we characterize here. The targeting of Ccp1 for selective H175 oxidation may be linked to the phosphorylation status of Y153 close to the heme since pY153 is abundant in certain proteoforms. In sum, when insufficient electrons from ferrocytochrome c are available to Ccp1 in mitochondria, hole hopping from its heme expands its physiological functions. Specifically, we observe an unprecedented hole-hopping sequence for heme labilization and identify hole-hopping pathways from the heme to novel substrates and to glutathione at Ccp1's surface. Furthermore, our results underscore the power of proteoform profiling by LC-MS/MS in exploring the cellular roles of oxidoreductases.