The role of UCP 1 in production of reactive oxygen species by mitochondria isolated from brown adipose tissue

We provide evidence that ablation or inhibition of, uncoupling protein 1 increases the rate of reactive oxygen containing species production by mitochondria from brown adipose tissue, no matter what electron transport chain substrate is used (succinate, glycerol-3-phosphate or pyruvate/malate). Consistent with these data are our observations that (a) the mitochondrial membrane potential is maximal when uncoupling protein 1 is ablated or inhibited and (b) oxygen consumption rates in mitochondria from uncoupling protein 1 knock-out mice, are significantly lower than those from wild-type mice, but equivalent to those from wild-type mice in the presence of GDP. In summary, we show that uncoupling protein 1 can affect reactive oxygen containing species production by isolated mitochondria from brown adipose tissue.     

Effects of running training on in vitro brown adipose tissue thermogenesis in rats

Brown adipose tissue (BAT) is a major site of nonshivering thermogenesis (NST) during cold acclimation for most mammals. Repetitive nonthermal stress such as immobilization has been shown to enhance the capacity of NST as cold acclimation. In the present study, the effects of running training, another type of nonthermal stress, were investigated on in vitro thermogenesis and the cellularity of interscapular BAT in rats. The rats were subjected to treadmill running for 30 min daily at 30 m/min under 8° inclination for 4–5 weeks. In vitro thermogenesis was then measured in minced tissue blocks incubated in a Krebs-Ringer phosphate buffer containing glucose and albumin at 37° C, using a Clark type oxygen electrode. The trained rats showed less body weight gain during the experiment. The weights of BAT and epididymal white adipose tissue were smaller in the trained rats. Noradrenaline- and glucagon-stimulated oxygen consumption were also significantly smaller in the trained rats. The tissue DNA level was greater in the trained rats, but the DNA content per tissue pad did not significantly differ. The results indicate that running training reduces BAT thermogenesis, possibly as an adaptation to conserve energy substrates for physical work.     

cAMP-dependent protein kinase from brown adipose tissue: temperature effects on kinetic properties and enzyme role in hibernating ground squirrels

Arousal from hibernation requires thermogenesis in brown adipose tissue, a process that is stimulated by β-adrenergic signals, leading to a rise in intracellular 3′,5′-cyclic adenosine monophosphate AMP (cAMP) and activating cAMP-dependent protein kinase A (PKA) to phosphorylate a suite of target proteins and activate lipolysis and uncoupled respiration. To determine whether specific adaptations (perhaps temperature-dependent) facilitate PKA kinetic properties or protein-phosphorylating ability, the catalytic subunit of PKA (PKAc) from interscapular brown adipose of the ground squirrel Spermophilus richardsonii, was purified (final specific activity = 279 nmol phosphate transferred per min per mg protein) and characterized. Physical properties of PKAc included a molecular weight of 41 kDa and an isoelectric point of 7.8 ± 0.08. A change in assay temperature from a euthermic value (37 °C) to one typical of hibernating body temperature (5 °C) had numerous significant effects on ground squirrel PKAc including: (a) pH optimum rose from 6.8 at 37 °C to 8.7 at 5 °C, (b) K_m values at 37 °C for Mg.ATP (49.2±3.4 M) and for two phosphate acceptors, Kemptide (50.0±5.5 M) and Histone IIA (0.41 ± 0.05 mg/ml) decreased by 53%, 80% and 51%, respectively, at 5 °C, and (c) inhibition by KCl, NaCl and NH₄Cl was reduced. However, temperature change had little or no effect on K_m values of rabbit PKAc, suggesting a specific positive thermal modulation of the hibernator enzyme. Arrhenius plots also differed for the two enzymes; ground squirrel PKAc showed a break in the Arrhenius relationship at 9 °C and activation energies that were 29.1 ± 1.0 kJ/mol for temperatures >9 °C and 2.3-fold higher at 68.1 ± 2.1 kJ/mol for temperatures <9 °C, whereas the rabbit enzyme showed a breakpoint at 17 °C with a 13-fold higher activation energy over the lower temperature range. However, fluorescence analysis of PKAc in the absence of substrates, showed a linear change in fluorescence intensity and wavelength of maximal fluorescence over the entire temperature range; this suggested that the protein conformational change indicated by the break in the Arrhenius plot was substrate-related. Temperature change also affected the Hill coefficient for cAMP dissociation of the ground squirrel PKA holoenzyme which rose from 1.12 ± 0.18 at 37 °C to 2.19 ± 0.07 at 5 °C, making the release of catalytic subunits at low temperature much more responsive to small changes in cAMP levels. Analysis of PKAc function via in vitro incubations of extracts of ground squirrel brown adipose with ³²P-ATP + cAMP in the presence versus absence of a PKA inhibitor, also revealed major differences in the patterns of phosphoproteins, both between euthermic and hibernating animals as well as between 37 and 5 °C incubation temperatures; this suggests that there are both different targets of PKAc phosphorylation in the hibernating animal and that temperature affects the capacity of PKAc to phosphorylate different targets. Both of these observations, plus the species-specific and temperature-dependent changes in ground squirrel PKAc kinetic properties, suggest differential control of the enzyme in vivo at euthermic versus hibernating body temperatures in a manner that would facilitate a rapid and large activation of the enzyme during arousal from torpor. Accepted: 10 July 1998     

GDF1 is a novel mediator of macrophage infiltration in brown adipose tissue of obese mice

We previously demonstrated a marked upregulation in the bone morphogenic protein (BMP)/growth differentiation factor (GDF) family member, GDF5, which is capable of promoting brown adipogenesis, in brown adipose tissue (BAT) of obese mice. In this study, we identified other GDF family members, besides GDF5 that are responsive to different obesogenic signals in BAT using inborn and acquired obesity animal models. In BAT from leptin-deficient ob/ob mice, GDF1 expression was preferentially downregulated, whereas the expression of several other genes in the BMP/GDF family, including GDF5, was upregulated. Moreover, in cultured brown adipocytes exposed to tunicamycin and hydrogen peroxide, at concentrations not affecting cellular viability, GDF1 expression was significantly downregulated. Recombinant GDF1 failed to significantly alter brown adipogenesis, despite the promoted phosphorylation of Smad1/5/8 in cultured brown adipocytes, but accelerated Smad1/5/8 phosphorylation with a concomitant increase in the number of migrating cells during exposure in a manner sensitive to activin-like kinase inhibitors in macrophagic RAW264.7 cells. Similarly, accelerated migration was observed in murine peritoneal macrophages exposed to GDF1. These results indicate that obesity could lead to predominant downregulation of GDF1 expression in BAT, which can modulate cellular migration through a mechanism relevant to activation of the downstream Smad signaling pathway in adjacent macrophages.     

Simultaneous detection of NOS-3 protein expression and nitric oxide production using a flow cytometer

Nitric oxide (NO) is involved in the regulation of SMC proliferation during intimal hyperplasia as has been shown by the inhibitory effect on intimal hyperplasia of adenovirus-mediated ceNOS overexpression in injured arteries in pig. Good assays to quantify the NO-producing enzymes, i.e., NO synthases (NOS), are essential to analyze the mechanism of action of NO in this process. We have developed novel flow cytometric assays for the simultaneous detection of NOS-3 protein, using NOS-3 specific antibodies, and NO production using 4,5-diaminofluorescein-diacetate (DAF-2/DA). The presence of NOS-3 protein and NO production is demonstrated on human A549 and HepG2 cells infected with a NOS-3 adenovirus (Ad.NOS-3). A comparative study showed that the flow cytometric assays are equally sensitive as Western blot analysis, the citrulline assay, or the Sievers assay. On human endothelial and SMC, NOS-3 protein and NO production were simultaneously detected with the assays, both under basal conditions and after Ad.NOS-3transduction. Simultaneous analysis of NOS-3 protein and NO production, made possible by the here-described novel flow cytometric assays, is of significant value to those investigating NOS-3 and NO.     

Correlation of exhaled breath temperature with bronchial blood flow in asthma

In asthma elevated rates of exhaled breath temperature changes (Δe°T) and bronchial blood flow (Q_aw) may be due to increased vascularity of the airway mucosa as a result of inflammation.We investigated the relationship of Δe°T with Q_aw and airway inflammation as assessed by exhaled nitric oxide (NO). We also studied the anti-inflammatory and vasoactive effects of inhaled corticosteroid and β₂-agonist.Δe°T was confirmed to be elevated (7.27 ± 0.6 Δ°C/s) in 19 asthmatic subjects (mean age ± SEM, 40 ± 6 yr; 6 male, FEV₁ 74 ± 6 % predicted) compared to 16 normal volunteers (4.23 ± 0.41 Δ°C/s, p < 0.01) (30 ± 2 yr) and was significantly increased after salbutamol inhalation in normal subjects (7.8 ± 0.6 Δ°C/ s, p < 0.05) but not in asthmatic patients. Q_aw, measured using an acetylene dilution method was also elevated in patients with asthma compared to normal subjects (49.47 ± 2.06 and 31.56 ± 1.6 μl/ml/min p < 0.01) and correlated with exhaled NO (r = 0.57, p < 0.05) and Δe°T (r = 0.525, p < 0.05). In asthma patients, Q_aw was reduced 30 minutes after the inhalation of budesonide 400 μg (21.0 ± 2.3 μl/ml/min, p < 0.05) but was not affected by salbutamol.Δe°T correlates with Q_aw and exhaled NO in asthmatic patients and therefore may reflect airway inflammation, as confirmed by the rapid response to steroids.     

Interaction between heat acclimation and exogenous insulin in brown adipose tissue of rats

Seventy-one male Wistar strain rats (7 weeks old) were kept at 5, 25, or 34° C, respectively, for 2 weeks with or without insulin administration. Insulin (Novo Lente MC) was given subcutaneously in a dose of 3.62 nmol/125 µl saline per 100 g body weight. An apparent effect of insulin treatment was noted only in heat-exposed rats, resulting in a remarkable gain in inter-scapular brown adipose tissue (BAT) mass of heat-acclimated, insulin-treated rats in terms of weight or weight per unit body weight. The BAT from heat-acclimated, insulin-treated rats had significantly higher levels of protein, DNA, RNA, and triglyceride than BAT from heat-acclimated, saline-treated rats. Therefore, it seems likely that the growth of BAT in heat-acclimated, insulin-treated rats was mostly due to the anabolic effects of insulin. The uncoupling protein mRNA was, however, present in BAT of heat-acclimated, insulin-treated rats at rather a depressed level, explaining a corresponding decrease in cold tolerance. On the other hand, the expression of insulin receptor mRNA was attenuated in BAT of rats from all the insulin-treated groups, possibly due to the down-regulation of insulin. Thus, there appeared to be some linkage among BAT, heat acclimation, and insulin.     

Modulation of beta-oxidation and proton conductance pathway of brown adipose tissue in hypo- and hyperinsulinemic states

The metabolic capacity of interscapular brown adipose tissue of hypoinsulinemic (diabetic) rats is decreased and a reduced beta-oxidative capacity contributes to this metabolic alteration. It was thus of interest to compare, in diabetic and in chronically (8 days) insulin-infused rats, the beta-oxidative capacity and indices of the thermogenic state (GDP-binding and 32 000 Mr protein) in this tissue. Mitochondrial GDP-binding and 32 000 Mr protein were both decreased in diabetic rats compared to appropriate controls and markedly increased as was also the beta-oxidative capacity in hyperinsulinemic rats.     

Tea catechins enhance the mRNA expression of uncoupling protein 1 in rat brown adipose tissue

The aim of the present study was to determine whether the antiobesity effects of tea catechins (TCs) are associated with the expression of uncoupling protein 1 (UCP1) in brown adipose tissue (BAT). Male Sprague–Dawley rats were fed a high-fat (HF; 35% fat) diet for 5 weeks, then divided into four groups and fed an HF, HF with 0.5% TC (HFTC), normal-fat (NF; 5% fat) or NF with 0.5% TC (NFTC) diet for 8 weeks. At the end of the experimental period, perirenal and epididymal white adipose tissues (WATs) and interscapular BAT were isolated. The NFTC group had significantly lower perirenal WAT weights than the NF group (NF: 12.7±0.53 g; NFTC: 10.2±0.43 g; P<.01), but the HF and HFTC groups did not differ significantly. TC intake had no effects on epididymal WAT weights. The NFTC and HFTC groups had significantly lower BAT weights than the NF and HF groups, respectively. The NFTC group had significantly higher UCP1 mRNA levels in BAT than the NF group (NF: 0.35±0.02; NFTC: 0.60±0.11; P<.05), but the HF and HFTC groups did not differ significantly. Thus, TC intake in the context of the NF diet reduced perirenal WAT weight and up-regulated UCP1 mRNA expression in BAT. These results suggest that the suppressive effect of TC on body fat accumulation is associated with UCP1 expression in BAT.     

β‐amyloid decreases detectable endothelial nitric oxide synthase in human erythrocytes: a role for membrane acetylcholinesterase

Until few years ago, many studies of Alzheimer's disease investigated the effects of this syndrome in the central nervous system. Only recently, the detection of amyloid beta peptide (Aβ) in the blood has evidenced the necessity to extend studies on extraneuronal cells, particularly on erythrocytes. Aβ is also present in brain capillaries, where it interacts with the erythrocytes, inducing several metabolic and functional alterations. Recently, functionally active endothelial type nitric oxide synthase (eNOS) was discovered in human erythrocytes. The goal of the present study was to evidence the effect of Aβ on erythrocyte eNOS. We found that Aβ following to 24‐h exposure causes a decrease in the immune staining of erythrocyte eNOS. Concurrently, Aβ alters erythrocyte cell morphology, decreases nitrites and nitrates levels, and affects membrane acetylcholinesterase activity. Propidium, an acetylcholinesterase inhibitor, was able to reverse the effects elicited by Aβ. These events could contribute to the vascular alterations associated with Alzheimer's disease disease. Copyright © 2012 John Wiley & Sons, Ltd.     

The effects of meal-feeding and the diurnal cycle on lipogenesis in brown adipose tissue of rats

A significant diurnal variation in the rates of lipogenesisin vivo in brown adipose tissue occurred in both virgin and lactating rats. On a meal-feeding regime of either a chow, high-sucrose, or high-lipid diet, there was a very large increase in BAT lipogenesis following the meal. The rates observed after the sucrose meal are the highest so far reported. There was no significant difference in BAT lipogenesis between lactating and virgin rats, contrary to previous reports by others. The pattern of stimulation of BAT lipogenesis by these feeding regimes was different from that for white adipose tissue and liver and was not correlated with plasma insulin levels.     

Effect of cold on lipid peroxidation in the brown adipose tissue and liver of rats

1. Lipid peroxidation in the interscapular brown adipose tissue (iBAT) and liver was studied in rats acclimated to room (23±1 °C) and low temperature (5±1 °C, 42 days), as well as in animals exposed to 5±1 °C for 24 h; in addition, the tissue metallothionein (MT) and iron were determined.     

Monocyte chemoattractant protein-1 and nitric oxide promote adipogenesis in a model that mimics obesity

Objective: An increasing body of evidence is emerging linking adipogenesis and inflammation. Obesity, alone or as a part of the metabolic syndrome, is characterized by a state of chronic low‐level inflammation as revealed by raised plasma levels of inflammatory cytokines and acute‐phase proteins. If inflammation can, in turn, increase adipose tissue growth, this may be the basis for a positive feedback loop in obesity. We have developed a tissue engineering model for growing adipose tissue in the mouse that allows quantification of increases in adipogenesis. In this study, we evaluated the adipogenic potential of the inflammogens monocyte chemoattractant protein (MCP)‐1 and zymosan‐A (Zy) in a murine tissue engineering model. Research Methods and Procedures: MCP‐1 and Zy were added to chambers filled with Matrigel and fibroblast growth factor 2. To analyze the role of inducible nitric oxide synthase (iNOS), the iNOS inhibitor aminoguanidine was added to the chamber. Results: Our results show that MCP‐1 generated proportionally large quantities of new adipose tissue. This neoadipogenesis was accompanied by an ingrowth of macrophages and could be mimicked by Zy. Aminoguanidine significantly inhibited the formation of adipose tissue. Discussion: Our findings demonstrate that low‐grade inflammation and iNOS expression are important factors in adipogenesis. Because fat neoformation in obesity and the metabolic syndrome is believed to be mediated by macrophage‐derived proinflammatory cytokines, this adipose tissue engineering system provides a model that could potentially be used to further unravel the pathogenesis of these two metabolic disorders.     

Effect of lactoferrin on the production of tumor necrosis factor‐α and nitric oxide

One of the biological functions of lactoferrin is the modulation of the host defense systems, including cytokine production and immune responses. We have tested the effect of lactoferrin on the productions of tumor necrosis factor‐α and nitric oxide in some cells. Lactoferrin itself did not induce either tumor necrosis factor‐α production or nitric oxide production, but lipopolysaccharide‐stimulated tumor necrosis factor‐α production of macrophages and monocytes were inhibited by lactoferrin treatment combined with stimulant. The induction of nitric oxide synthesis in stimulated macrophages and vascular smooth muscle cells was not affected by the lactoferrin. J. Cell. Biochem. 76:30–36, 1999. © 1999 Wiley‐Liss, Inc.     

A literature review: the effects of magnetic field exposure on blood flow and blood vessels in the microvasculature

The effect of magnetic field (MF) exposure on microcirculation and microvasculature is not clear or widely explored. In the limited body of data that exists, there are contradictions as to the effects of MFs on blood perfusion and pressure. Approximately half of the cited studies indicate a vasodilatory effect of MFs; the remaining half indicate that MFs could trigger either vasodilation or vasoconstriction depending on initial vessel tone. Few studies indicate that MFs cause a decrease in perfusion or no effect. There is a further lack of investigation into the cellular effects of MFs on microcirculation and microvasculature. The role of nitric oxide (NO) in mediating microcirculatory MF effects has been minimally explored and results are mixed, with four studies supporting an increase in NO activity, one supporting a biphasic effect, and five indicating no effect. MF effects on angiogenesis are also reported: seven studies supporting an increase and two a decrease. Possible reasons for these contradictions are explored. This review also considers the effects of magnetic resonance imaging (MRI) and anesthetics on microcirculation. Recommendations for future work include studies aimed at the cellular/mechanistic level, studies involving perfusion measurements both during and post-exposure, studies testing the effect of MFs on anesthetics, and investigation into the microcirculatory effects of MRI.     

Effect ofE. coli endotoxin on temperature,oxygen consumption and brown adipose tissue thermogenesis in rats and mice

The effects ofE. coli endotoxin 0127 B8 on oxygen consumption, temperature, and on the activity of the proton conductance pathway in brown adipose tissue (BAT) were investigated in rats and mice. In rats an increase was observed in rectal and skin temperature, whole body oxygen consumption and GDP binding in BAT. In mice only the rise in rectal and skin temperature were significantly changed by endotoxin administration.These findings suggest that in some species BAT is involved in the production of endotoxin induced fever and increased energy expenditure.     

Cold-induced changes in brown adipose tissue thermogenic capacity of immunocompetent and immunodeficient hairless mice

Mild cold acclimation (22°C, 3 weeks) of hairless mice was shown to increase 5-fold the brown adipose tissue uncoupling protein content in immunodeficient BALB/c nu/nu mice, but by only 2.3-fold in immunocompetent BFU mice. The difference in activation of brown adipose tissue thermogenic capacity was due to a 2-fold increase in the content of brown adipose tissue in nu/nu mice only, which was paralleled by an increase in brown adipose tissue protein but not DNA content. Likewise, only in nu/nu mice the cold acclimation increased the reaction of natural killer cells in blood and peritoneal exudate with a shift from spleen to lymph nodes and increased the phagocytic index. The results indicate that the immune system may influence the defence against cold at the level of brown adipose tissue thermogenesis.Abbreviations AU arbitrary unit(s) - bw body weight - HEMA 2-hydromethyl-metacrylate copolymer - BAT brown adipose tissue - UCP uncoupling protein - ATPase mitochondrial F_oF₁-ATPsynthase - IL-1 interleukin 1 - TNF tumour necrosis factor - NK cells natural killer cells - T _a ambient temperature