Suppression Of Oxygen Consumption Research Articles

Thioesterase Superfamily Member 1 Undergoes Stimulus‐coupled Reorganization to Regulate Metabolic Activity in Brown Adipose Tissue

Thioesterase superfamily member 1 (Them1) is a long chain fatty acyl‐CoA thioesterase that is enriched in brown adipose tissue. Them1 is markedly upregulated by cold ambient temperatures, but functions to suppress energy expenditure. Consequently Them1−/− mice are highly resistant to diet‐induced obesity‐related disorders, most notably hepatic steatosis and insulin resistance. Although Them1 reduces fatty acid availability for b‐oxidation in mitochondria, how it limits thermogenesis remains undefined. Here we show that Them1 undergoes marked intracellular conformational changes after stimulation to regulate metabolic activity.MethodsCultured brown adipocytes, which do not constitutively express Them1, were transfected with full‐length Them1 fused to EGFP as well as an engineered ascorbate peroxidase (APEX2) fusion protein. EGFP was visualized by confocal microscopy in the presence or absence of norepinephrine (NE) stimulation to determine the 3‐D localization of Them1. APEX2 was used for high resolution correlative light and electron microscopy (CLEM). Cells were further transfected with Them1 phosphomimetic mutants at S15, 18, and 25 to evaluate the role of phosphorylation in Them1 organization. Rates of NE‐stimulated fatty acid oxidation were determined by using a Seahorse XFe96. The localization of Them1 in brown adipose tissue of mice was determined after injection of CL316,243, a β3 adrenoceptor agonist. The primary amino acid sequence of Them1 was subjected to multiple analyses to predict disordered regions, aggregation, and hydrophobicity.ResultsThem1 formed puncta that were localized near lipid droplets and mitochondria in cultured brown adipose cells. In response to NE, Them1 was phosphorylated, which resulted in a diffuse intracellular localization. This change in Them1 localization also occurred in brown adipose tissue after CL316,243 administration to mice. Puncta formation activated Them1, as evidenced by suppression of oxygen consumption. CLEM revealed that puncta are highly organized membraneless organelles, which typically form after a phase separation in cells. To support this possibility, Them1 contains one intrinsically disordered region at the N‐terminus that contains multiple interacting motifs, as is frequently found in phase‐separating proteins. Similarly, phosphorylation is known to disrupt aggregation after a phase transition, resulting in a diffuse localization.ConclusionsOur data establish that Them1 forms membraneless organelles (puncta) that function to suppress thermogenesis. Additionally, puncta are dispersed after phosphorylation at S15 to maximize fatty acid oxidation during a period of energy demand and to allow for the rapid suppression of thermogenesis once the demand no longer exists. We speculate that Them1 is an attractive molecular target in the management of non‐alcoholic fatty liver disease and other obesity‐related disorders.

Diet-induced obesity causes peripheral and central ghrelin resistance by promoting inflammation.

Ghrelin, a stomach-derived orexigenic peptide, transmits starvation signals to the hypothalamus via the vagus afferent nerve. Peripheral administration of ghrelin does not induce food intake in high fat diet (HFD)-induced obese mice. We investigated whether this ghrelin resistance was caused by dysfunction of the vagus afferent pathway. Administration (s.c.) of ghrelin did not induce food intake, suppression of oxygen consumption, electrical activity of the vagal afferent nerve, phosphorylation of ERK2 and AMP-activated protein kinase alpha in the nodose ganglion, or Fos expression in hypothalamic arcuate nucleus of mice fed a HFD for 12 weeks. Administration of anti-ghrelin IgG did not induce suppression of food intake in HFD-fed mice. Expression levels of ghrelin receptor mRNA in the nodose ganglion and hypothalamus of HFD-fed mice were reduced. Inflammatory responses, including upregulation of macrophage/microglia markers and inflammatory cytokines, occurred in the nodose ganglion and hypothalamus of HFD-fed mice. A HFD blunted ghrelin signaling in the nodose ganglion via a mechanism involving in situ activation of inflammation. These results indicate that ghrelin resistance in the obese state may be caused by dysregulation of ghrelin signaling via the vagal afferent.

Studies on the photosensitized reduction of resorufin and implications for the detection of oxidative stress with Amplex Red

The photosensitized reduction of resorufin (RSF), the fluorescent product of Amplex Red, was investigated using electron spin resonance (ESR), optical absorption/fluorescence, and oxygen consumption measurements. Anaerobic reaction of RSF in the presence of the electron donor reduced nicotinamide adenine dinucleotide (NADH) demonstrated that during visible light irradiation (λ>300nm), RSF underwent one-electron reduction to produce a semiquinoneimine-type anion radical (RSF• ‾) as demonstrated by direct ESR. Spin-trapping studies of incubations containing RSF, 5,5-dimethyl-1-pyrroline N-oxide (DMPO) and NADH demonstrated, under irradiation with visible light, the production of the superoxide dismutase (SOD)-sensitive DMPO/•OOH adduct. Both absorption and fluorescence spectra of RSF in the presence of NADH demonstrated that the RSF• ‾ was further reduced during irradiation with formation of its colorless dihydroquinoneimine form, dihydroresorufin (RSFH2). Both RSF• ‾ and RSFH2, when formed in an aerobic system, were immediately oxidized by oxygen, which regenerated the dye and formed superoxide. Oxygen consumption measurements with a Clark-type oxygen electrode showed that molecular oxygen was consumed in a light-dependent process. The suppression of oxygen consumption by addition of SOD or catalase further confirmed the production of superoxide and hydrogen peroxide.

Modulation of platelet activating factor-induced glycogenolysis in the perfused rat liver after administration of endotoxin in vivo.

The effect of endotoxin treatment in vivo on platelet activating factor (PAF)-induced glycogenolysis was studied in the perfused rat liver. The addition of PAF (20 nM) to the perfusate increased glucose production concomitant with suppression of oxygen consumption in control rats without endotoxin treatment. At 6 h after endotoxin administration, PAF caused severe suppression of oxygen consumption, but glucose production was greatly inhibited. At 24 h after endotoxin treatment, PAF caused less suppression of oxygen consumption than the control, and glucose production was partially restored. The metabolic responses in the control rat were abolished by the simultaneous presence of cyclooxygenase- and lipoxygenase-inhibitors. Combined use of leukotriene (LT) D4- and thromboxane (Tx) A2-receptor antagonists inhibited the metabolic responses in the rat given endotoxin 6 h before. The efflux of Tx B2 during PAF-infusion decreased 24 h after endotoxin treatment, and Tx A2 receptor antagonist, but not LT D4 receptor antagonist, prevented the suppression of oxygen consumption. These results suggest that different eicosanoids are involved in PAF-induced glycogenolysis in different stages of endotoxemia, and that LT D1 may also play a role in PAF-induced glycogenolysis.

Hepatic clearance and retention of aluminium: studies in the isolated perfused rat liver

Aluminium (Al) exposure can result in Al accumulation in the liver and this metal can be toxic to the hepatic tissue at high concentrations. In the present study the model of the isolated perfused rat liver was used to investigate the hepatic handling of Al. Livers from male Wistar rats were perfused in a recirculating system for 240 min. The liver function remained unchanged at perfusate concentrations of Al ranging from 4.9 to 1530.0 μ/1. At higher Al levels of 6535.3–16 694.9 μ/1 signs of toxicity towards isolated perfused livers were observed as indicated by an increased release of the enzymes AST and ALT into the perfusate, a pronounced reduction of bile flow rate and a 50% suppression of oxygen consumption. The hepatic Al clearance was low and decreased with increasing concentrations of Al in the perfusate from 4.3 + 0.6 μ/min per g liver at a nominal Al concentration of 9.1 μ/l in control perfusate to 0.04 ± 0.02μl/min per g liver at the highest concentration group. There was almost a linear dose dependent retention of Al in the liver with 4.9–635.7 μ Al/1 perfusate while at higher concentrations Al levels in this organ increased disproportionally. It is concluded that by using the isolated perfused rat changes of liver functions occur only at very high Al concentrations in the perfusate and that only negligible amounts of Al are eliminated by the liver.

Phorbol ester, but not endotoxin, desensitizes mannan-induced glycogenolysis in the perfused rat liver.

Mannan, a ligand for the mannose/N-acetylglucosamine (GlcNAc) receptor, induces suppression of oxygen consumption and increases glucose production in the perfused rat liver, and repeated infusion of mannan causes desensitization of the responses. In this study, we examined whether activation of Kupffer cells by endotoxin and phorbol ester alters the glycogenolytic responses to mannan. Infusion of lipopolysaccharide (LPS, 10 micrograms/ ml) in the perfusate failed to inhibit the responses to mannan. Intravenous administration of LPS (1 mg/kg) 6 and 24 h before perfusion did not desensitize the responses to mannan, suggesting that the responses through mannose/GlcNAc receptors in the liver are retained even after activation of Kupffer cells by LPS. In contrast, prior infusion of phorbol 12-myristate 13-acetate (PMA, 100 nM) in vitro abolished the glycogenolytic responses to subsequently infused mannan, but not that to norepinephrine (100 nM), while prior infusions of 4-alpha-phorbol 12,13-didecanoate (100 nM), A23187 (50 nM), or forskolin (1 microM) had no effect on the mannan-induced responses. H-7, an inhibitor of protein kinase C, reduced the glycogenolytic responses to mannan, while it failed to restore the desensitization. These results suggest that protein kinase C may be involved in the process of glycogenolysis by mannan, but is unlikely to be involved in the homologous desensitization of the responses.

Partially deacetylated chitin and hyaluronan induce glycogenolysis in perfused rat liver.

Mannan interacts with mannose/N-acetylglucosamine (GlcNAc) receptors on the surface of both Kupffer cells and endothelial cells in the liver, and induces glycogenolysis through production of peptide-leukotriene (LT) in the perfused rat liver. In the present study, we examined whether positively and negatively charged GlcNAc-containing polysaccharides stimulate glycogenolysis in perfused rat liver. Infusion of the former, 67% deacetylated chitin (DAC), induced biphasic increases in glucose production and a steep decrease in oxygen consumption by the liver. ONO-1078, an LT D4 receptor antagonist, abolished the suppression of oxygen consumption and reduced the glucose production by DAC. Infusion of the latter, hyaluronan, stimulated glucose production with a concomitant increase in oxygen consumption. Ibuprofen, a cyclooxygenase inhibitor, reduced the glucose production by hyaluronan. Sequential infusions of mannan and DAC, but not hyaluronan, did not induce glycogenolytic responses when mannan was infused 20 min before the second stimulation. These results suggest that DAC, but not hyaluronan, stimulates mannose/GlcNAc receptors in the perfused rat liver, and that potent immunological activity induced by DAC may be mediated by activation of the receptors.

CHO cell responses to low oxygen: Regulation of oxygen consumption and sensitization to oxidative stress

We have investigated the regulation of oxygen consumption and modulation of glutathione levels in CHO-K1 cells under oxygen-limiting conditions. We report here suppression of oxygen consumption and alteration of the supply-dependent relationship as a consequence of prolonged hypoxic or anoxic exposure. The suppression is characterized by an increase in the value of P(o(2)/50) (the oxygen tension at which oxygen consumption is half maximal). Under prolonged anoxia there is also a decrease in the cells' potential to use oxygen. Elevated glucose consumption under low oxygen conditions may contribute to the suppression in respiration. The glutathione concentration remains constant throughout hypoxic exposure but may decrease by as much as 40% under anoxia. The glutathione level in hypoxic and anoxic cells increases by two- and four-fold, respectively, over that of the control cells when exposed to a cytotoxic level of oxygen (93%). This suggests that anoxic and hypoxic exposure sensitizes CHO cells to oxidative stress.

Enhanced copper toxicity resulting from environmental stress factor synergies

1. Circulatory and respiratory responses of Carcinus maenas to various combinations of salinity stress, progressive hypoxia and copper pollution (10 mg/l) have been investigated. 2. Salinity stress (8%.) was associated with increased heart rate and oxygen consumption. In contrast, simultaneous exposure to salinity stress and copper ions resulted in bradycardia and a progressive reduction in aerobic metabolism. 3. Hypoxia evoked bradycardia and suppression of oxygen consumption below a critical oxygen tension ( P c) in the range 66–80 mmHg. Similar responses were exhibited when copper ions were present although perfusion indices ( Q/ V o2) were elevated. 4. During simultaneous exposure to all three stress factors responses to hypoxia predominated. 5. Environmental stress factors synergistically enhanced copper toxicity resulting in ca 40% mortality with copper + salinity stress, 100% mortality with copper + hypoxic stress and 100% mortality together with reduction of time to death with copper + hypoxia + salinity stress.

Inhibition of human platelet lipoxygenase by cyanide

Potassium cyanide inhibited the lipoxygenase activity of a human platelet cytosolic fraction in a concentration-dependent manner (ID50=2 mM). The inhibition was monitored by spectrophotometry (conjugation of diene bonds at 236 nm), by chromatography (inhibition of formation of 12-hydroperoxy eicosatetraenoic acid) as well as by measuring suppression of oxygen consumption. The lipoxygenase activity of intact platelets was also inhibited by KCN as evidenced by the reduction in 12-hydroxy-eicosatetraenoic acid formation in response to thrombin.