Interactions between an alpha2-adrenergic antagonist and a beta3-adrenergic agonist on the expression of UCP2 and UCP3 in rats

This experimental trial was devised to assess whether selective beta3-adrenergic receptor (AR) stimulation and simultaneous blockade of alpha2-AR would affect thermoregulation. With this purpose, the individual and combined administration of a beta-AR agonist, trecadrine, and an alpha2-AR antagonist, yohimbine, were evaluated. Yohimbine produced a marked decrease (p < 0.001) in body temperature one hour after administration (5 mg kg(-1), i.p.) and blocked the thermogenic effect of trecadrine (1 mg kg(-1), i.p.) when simultaneously administered. Uncoupling protein-2 expression in skeletal muscle was downregulated (p < 0.05) by trecadrine, while yohimbine had no effect. White adipose tissue UCP2 and muscle UCP3 were not modified by either trecadrine or yohimbine administration. Liver UCP2 mRNA expression was significantly decreased by yohimbine (p < 0.05). However, this downregulation does not seem to explain the reduction in temperature produced by yohimbine given the fact that trecadrine produced a similar downregulation of hepatic UCP2 (p < 0.05). The present work indicates that alpha2-AR antagonism blocks the thermogenic effects mediated by beta3-AR stimulation, contrary to our expectations, suggesting a possible interplay between both mechanisms. Moreover, these effects are not apparently explained by changes in UCP2 and UCP3.     

Links between fatty acids and expression of UCP2 and UCP3 mRNAs

Physiological and pathological states that are associated with elevated plasma fatty acids (FAs) increase uncoupling protein 2 (UCP2) mRNA in white adipose tissue and UCP3 mRNA in skeletal muscle and heart. A direct effect of unsaturated fatty acids from all classes has been shown in various cultured cells. There is evidence that FAs could induce expression of UCPs by acting as ligands for peroxisome proliferator-activated receptors, influencing the function of sterol responsive element binding protein or activating 5'-AMP-activated protein kinase. Oleic acid has been shown to stimulate the activity of the promoter regions of UCP2 and UCP3 genes and the FA responsive regions are beginning to be characterised.     

An interplay between UCP2 and ROS protects cells from high-salt-induced injury through autophagy stimulation

The mitochondrial uncoupling protein 2 (UCP2) plays a protective function in the vascular disease of both animal models and humans. UCP2 downregulation upon high-salt feeding favors vascular dysfunction in knock-out mice, and accelerates cerebrovascular and renal damage in the stroke-prone spontaneously hypertensive rat. Overexpression of UCP2 counteracts the negative effects of high-salt feeding in both animal models. We tested in vitro the ability of UCP2 to stimulate autophagy and mitophagy as a mechanism mediating its protective effects upon high-salt exposure in endothelial and renal tubular cells. UCP2 silencing reduced autophagy and mitophagy, whereas the opposite was true upon UCP2 overexpression. High-salt exposure increased level of reactive oxygen species (ROS), UCP2, autophagy and autophagic flux in both endothelial and renal tubular cells. In contrast, high-salt was unable to induce autophagy and autophagic flux in UCP2-silenced cells, concomitantly with excessive ROS accumulation. The addition of an autophagy inducer, Tat-Beclin 1, rescued the viability of UCP2-silenced cells even when exposed to high-salt. In summary, UCP2 mediated the interaction between high-salt-induced oxidative stress and autophagy to preserve viability of both endothelial and renal tubular cells. In the presence of excessive ROS accumulation (achieved upon UCP2 silencing and high-salt exposure of silenced cells) autophagy was turned off. In this condition, an exogenous autophagy inducer rescued the cellular damage induced by excess ROS level. Our data confirm the protective role of UCP2 toward high-salt-induced vascular and renal injury, and they underscore the role of autophagy/mitophagy as a mechanism counteracting the high-salt-induced oxidative stress damage.Subject terms: Macroautophagy, Macroautophagy     

Differential regulation of the promoter activity of the mouse UCP2 and UCP3 genes by MyoD and myogenin

UCP2 and UCP3 are members of the uncoupling protein family, which may play roles in energy homeostasis. In order to determine the regulation of the predominant expression of UCP3 in skeletal muscle, the effects of differentiation and myogenic regulatory factors on the promoter activities of the mouse UCP2 and UCP3 genes were studied. Reporter plasmids, containing approximately 3 kb of the 5'-upstream region of the mouse UCP2 and UCP3 genes, were transfected into C2C12 myoblasts, which were then induced to differentiate. Differentiation positively induced the reporter expression about 20-fold via the UCP3 promoter, but by only 2-fold via the UCP2 promoter. C2C12 myoblasts were cotransfected with expression vectors for myogenin and/or MyoD as well as reporter constructs. The simultaneous expression of myogenin and MyoD caused an additional 20-fold increase in the reporter expression via the UCP3 promoter, but only a weak effect via the UCP2 promoter. In L6 myoblasts, only MyoD activated the UCP3 promoter, but in 3T3-L1 cells neither factor activated the UCP3 promoter, indicating that additional cofactors are required, which are present only in C2C12 myoblasts. The expression of UCP2 and UCP3 is differentially regulated during muscle differentiation due to the different responsiveness of their promoter regions to myogenin and MyoD.     

Physiological regulation of the transport activity in the uncoupling proteins UCP1 and UCP2

Brown fat is a thermogenic organ that allows newborns and small mammals to maintain a stable body temperature when exposed to cold. The heat generation capacity is based on the uncoupling of respiration from ATP synthesis mediated by the uncoupling protein UCP1. The first studies on the properties of these mitochondria revealed that fatty acid removal was an absolute prerequisite for respiratory control. Thus fatty acids, that are substrate for oxidation, were proposed as regulators of respiration. However, their ability to uncouple all types of mitochondria and the demonstration that several mitochondrial carriers catalyze the translocation of the fatty acid anion have made them unlikely candidates for a specific role in brown fat. Nevertheless, data strongly argue for a physiological function. First, fatty acids mimic the noradrenaline effects on adipocytes. Second, there exists a precise correlation between fatty acid sensitivity and the levels of UCP1. Finally, fatty acids increase the conductance by facilitating proton translocation, a mechanism that is distinct from the fatty acid uncoupling mediated by other mitochondrial carriers. The regulation of UCP1 and UCP2 by retinoids and the lack of effects of fatty acids on UCP2 or UCP3 are starting to set differences among the new uncoupling proteins.     

Site-specific interplay between O-GlcNAcylation and phosphorylation in cellular regulation

Ser(Thr)-O-linked β-N-acetylglucosamine (O-GlcNAc) is a ubiquitous modification of nucleocytoplasmic proteins. Extensive crosstalk exists between O-GlcNAcylation and phosphorylation, which regulates signaling in response to nutrients/stress. The development of novel O-GlcNAc detection and enrichment methods has improved our understanding of O-GlcNAc functions. Mass spectrometry has revealed O-GlcNAc’s many interactions with phosphorylation-mediated signaling. However, mechanisms regulating O-GlcNAcylation and phosphorylation are quite different. Phosphorylation is catalyzed by hundreds of distinct kinases. In contrast, in mammals, uridine diphospho-N-acetylglucosamine:polypeptide β-N-acetylglucosaminyl transferase (OGT) and β-D-N-acetylglucosaminidase (OGA) are encoded by single highly conserved genes. Both OGT’s and OGA’s specificities are determined by their transient associations with many other proteins to create a multitude of specific holoenzymes. The extensive crosstalk between O-GlcNAcylation and phosphorylation represents a new paradigm for cellular signaling.     

Up-regulation of uterine UCP2 and UCP3 in pregnant rats.

Pregnancy produces profound changes in hormone dynamics, thermoregulation and energy metabolism. Uncoupling proteins (UCPs) have been identified in a variety of tissues and UCP1 is known to play important roles in energy homeostasis, while the regulation of UCP2 and UCP3 is still unclear. The present study aimed to investigate the effects of the changes during pregnancy on UCP gene expression in the uterus, as well as in brown adipose tissue (BAT), white adipose tissue (WAT), soleus muscle (Muscle), and liver, throughout the estrus and metestrus periods, at early, middle and late stages in pregnancy, and during post-gestational stages. The expression of uterine UCP2 and UCP3 were up-regulated by 3.2- and 1. 5-fold, respectively, during the late stage of pregnancy with an increase of WAT leptin mRNA expression and exogenous administration of leptin resulted in induction of the uterine UCP2 and UCP3 levels. Contrary to uterine UCPs, UCP1 mRNA expression in BAT was down-regulated by 0.5-fold and there were no remarkable changes in WAT or liver UCP2, or Muscle UCP3 expression throughout the periods. These results indicate that UCP gene expressions during pregnancy are regulated tissue-dependently, and up-regulation of uterine UCP2 and UCP3 mRNA may be due to increased leptin levels.     

Synaptic relationships between proopiomelanocortin- and ghrelin-containing neurons in the rat arcuate nucleus

Both proopiomelanocortin (POMC) and ghrelin peptides are implicated in the feeding regulation. The synaptic relationships between POMC- and ghrelin-containing neurons in the hypothalamic arcuate nucleus were studied using double-immunostaining methods at the light and electron microscope levels. Many POMC-like immunoreactive axon terminals were found to be apposed to ghrelin-like immunoreactive neurons and also to make synapses with ghrelin-like immunoreactive neuronal perikarya and dendritic processes. Most of the synapses were symmetrical in shape. A small number of synapses made by ghrelin-like immunoreactive axon terminals on POMC-like immunoreactive neurons were also identified. Both the POMC- and ghrelin-like immunoreactive neurons were found to contain large dense granular vesicles. These data suggest that the POMC-producing neurons are modulated via synaptic communication with ghrelin-containing neurons. Moreover, ghrelin-containing neurons may also have a feedback effect on POMC-containing neurons through direct synaptic contacts.     

黄体酮对3T3-L1脂肪前体细胞成脂分化后细胞中UCP2基因表达的影响

目的:观察体外培养条件下3T3-L1脂肪前体细胞诱导分化成的成熟脂肪细胞中解偶联蛋白2(UCP2)mRNA表达水平及黄体酮对其表达的影响。方法:体外培养3T3-L1脂肪细胞,在诱导3T3-L1脂肪细胞分化成熟后,经不同黄体酮浓度10μm/25μM/50μM/75μM/100μM刺激后,抽提总RNA,用RT-PCR检测UCP2 mRNA的表达。结果:黄体酮会促进成熟脂肪细胞中UCP2 mRNA的表达,(P<0.05)其中25μM浓度刺激下UCP2 mRNA表达量最高。结论:体外培养中,黄体酮对成熟脂肪细胞中UCP2 mRNA的表达与调控具有一定的影响。     

黄体酮对3T3-L1脂肪前体细胞成脂分化后细胞中UCP2基因表达的影响

目的：观察体外培养条件下3T3-L1脂肪前体细胞诱导分化成的成熟脂肪细胞中解偶联蛋白2（UCP2）mRNA表达水平及黄体酮对其表达的影响。方法：体外培养3T3-L1脂肪细胞,在诱导3T3．L1脂肪细胞分化成熟后,经不同黄体酮浓度10μm／25μM／50μM／75μM/100μM刺激后,抽提总RNA,用RT—PCR检测UCP2mRNA的表达。结果：黄体酮会促进成熟脂肪细胞中UCP2mRNA的表达,（P〈0．05）其中25μM浓度刺激下UCP2mRNA表达量最高。结论：体外培养中,黄体酮对成熟脂肪细胞中UCP2mRNA的表达与调控具有一定的影响。     

Associations between UCP1 -3826A/G,UCP2 -866G/A,Ala55Val and Ins/Del,and UCP3 -55C/T Polymorphisms and Susceptibility to Type 2 Diabetes Mellitus: Case-Control Study and Meta-Analysis

Background

Some studies have reported associations between five uncoupling protein (UCP) 1–3 polymorphisms and type 2 diabetes mellitus (T2DM). However, other studies have failed to confirm the associations. This paper describes a case-control study and a meta-analysis conducted to attempt to determine whether the following polymorphisms are associated with T2DM: -3826A/G (UCP1); -866G/A, Ala55Val and Ins/Del (UCP2) and -55C/T (UCP3).

Methods

The case-control study enrolled 981 T2DM patients and 534 nondiabetic subjects, all of European ancestry. A literature search was run to identify all studies that investigated associations between UCP1–3 polymorphisms and T2DM. Pooled odds ratios (OR) were calculated for allele contrast, additive, recessive, dominant and co-dominant inheritance models. Sensitivity analyses were performed after stratification by ethnicity.

Results

In the case-control study the frequencies of the UCP polymorphisms did not differ significantly between T2DM and nondiabetic groups (P>0.05). Twenty-three studies were eligible for the meta-analysis. Meta-analysis results showed that the Ala55Val polymorphism was associated with T2DM under a dominant model (OR = 1.27, 95% CI 1.03–1.57); while the -55C/T polymorphism was associated with this disease in almost all genetic models: allele contrast (OR = 1.17, 95% CI 1.02–1.34), additive (OR = 1.32, 95% CI 1.01–1.72) and dominant (OR = 1.18, 95% CI 1.02–1.37). However, after stratification by ethnicity, the UCP2 55Val and UCP3 -55C/T alleles remained associated with T2DM only in Asians (OR = 1.25, 95% CI 1.02–1.51 and OR = 1.22, 95% CI 1.04–1.44, respectively; allele contrast model). No significant association of the -3826A/G, -866G/A and Ins/Del polymorphisms with T2DM was observed.

Conclusions

In our case-control study of people with European ancestry we were not able to demonstrate any association between the UCP polymorphisms and T2DM; however, our meta-analysis detected a significant association between the UCP2 Ala55Val and UCP3 -55C/T polymorphisms and increased susceptibility for T2DM in Asians.     

Morphological interaction between galanin-like peptide- and dopamine-containing neurons in the rat arcuate nucleus

Galanin-like peptide (GALP) is a 60-amino acid neuropeptide that plays an important role in the neuronal regulation of feeding, energy balance and reproduction. GALP is produced in the hypothalamic arcuate nucleus, an area containing, amongst other neuron types, two populations of neurons in which we were interested: a population of GALP-containing neurons which regulate energy balance and reproduction, and a second population consisting of tuberoinfundibular dopaminergic neurons which suppress prolactin secretion from the adenohypophysis. To characterize morphologically the relationship between GALP and dopamine-containing neurons in the arcuate nucleus, a double immunofluorescence study was performed on cryosections from rat brain. Immunohistochemical double labeling studies revealed that GALP-immunoreactive nerve fibers made direct contact on tyrosine hydroxylase (TH)-containing neuronal cell bodies in the arcuate nucleus. These results suggest that GALP-containing neurons innervate tuberoinfundibular dopaminergic neurons.     

Synaptic interactions between ghrelin- and neuropeptide Y-containing neurons in the rat arcuate nucleus

Morphological relationships between neuropeptide Y- (NPY) like and ghrelin-like immunoreactive neurons in the arcuate nucleus (ARC) were examined using light and electron microscopy techniques. At the light microscope level, both neuron types were found distributed in the ARC and could be observed making contact with each other. Using a preembedding double immunostaining technique, some NPY-immunoreactive axon terminals were observed at the electron microscope level to make synapses on ghrelin-immunoreactive cell bodies and dendrites. While the axo-somatic synapses were mostly symmetric in nature, the axo-dendritic synapses were both symmetric and asymmetric. In contrast, ghrelin-like immunoreactive (ghrelin-LI) axon terminals were found to make synapses on NPY-like immunoreactive (NPY-LI) dendrites although no NPY-like immunoreactive perikarya were identified receiving synapses from ghrelin-LI axon terminals. NPY-like axon terminals were also found making synapses on NPY-like neurons. Axo-axonic synapses were also identified between NPY- and ghrelin-like axon terminals. The present study shows that NPY- and ghrelin-LI neurons could influence each other by synaptic transmission through axo-somatic, axo-dendritic and even axo-axonic synapses, and suggests that they participate in a common effort to regulate the food-intake behavior through complex synaptic relationships.     

中枢β—内啡肽和5—羟色胺在低氧通气调控中的关系

实验在去双侧外周化学感受器传入的麻醉雄性家兔上进行。给动物吸低氧混合气(10%O_2于N_2中)导致通气抑制后,从动物侧脑室注入β-内啡肽拮抗剂纳洛酮使抑制的通气被“翻转”;注入拟似剂羟甲芬太尼则使抑制更加深化。此时中枢5-羟色胺含量均较给药前明显升高。给动物注入外源性5-羟色胺后,中枢β-内啡肽含量较给药前明显下降,此时通气回复至对照水平。结果提示:中枢β-内啡肽系统是引起低氧通气抑制的基本和直接因素,而5-羟色胺系统则是作为一种神经调质,通过调制其β-内啡肽活动而间接对低氧通气反应产生影响。     

Relationship between insulin stimulation and endogenous regulation of 2-deoxyglucose uptake in 3T3-L1 adipocytes

The occurrence of the endogenous regulatory response to high rates of 2-deoxyglucose (2-DG) uptake, as previously described for C6 glioma cells during incubation with 2 mM 2-DG (Lange et al.: J. Cell. Physiol., 1989), was studied in 3T3-L1 preadipocytes and adipocytes, and the influence of insulin on this endogenous uptake regulation was examined. In contrast to 3T3-L1 preadipocytes, insulin-sensitive differentiated 3T3-L1 adipocytes displayed the time-dependent cyclic pattern of 2-DG uptake rates characteristic of the membrane-limited and endogenously regulated cellular state of hexose utilization. Although insulin induced a threefold stimulation of 2-DG tracer uptake in adipocytes, the hormone did not additionally stimulate the uptake rates or affect the periodic response: maximum and minimum levels of uptake remained unchanged. Scanning electron microscopy (SEM) revealed that the acquirement of the differentiated state is accompanied by a conspicuous transformation of the smooth surface of undifferentiated 3T3-L1 cells into a surface covered by numerous microvilli of uniform size and appearance. Treatment with insulin (10 mU/ml; 10 minutes) converted these microvilli into voluminous saccular membrane protrusions of the same type as had been formed during incubation of 3T3-L1 adipocytes with 2 mM 2-DG, and which have previously been shown to be involved in the endogenous uptake regulation of C6 glioma cells (Lange et al.: J. Cell. Physiol., 1989). These insulin-induced saccated membrane areas appeared to become integrated into the cell surface. Accordingly, insulin treatment caused a twofold increase of the intracellular distribution space of 3-O-methylglucose (3-OMG) in 3T3-L1 adipocytes. This insulin-induced increase of the 3-OMG distribution space exhibited the same time (t1/2 = 2-2.5 minutes) and dose dependence (EC50 = 20 nM) as the insulin-induced stimulation of 3-OMG transport. Glucose deprivation during the differentiation period inhibited the outgrowth of microvilli from the cell surface. Glucose starvation (18 hours at less than 0.5 mM) induced a conspicuous reduction of the length of microvilli on differentiated 3T3-L1 cells. In this state, the stalks of the microvilli are almost invisible and the enlarged spherical tips of the microvilli (with an average diameter of 370 nm compared to 230 nm of fed cells) appeared to protrude directly out of the cell surface. Starvation-induced shortening of microvilli was accompanied by a threefold increase of the basal 3-OMG transport rate and a greater than twofold increase of the intracellular 3-OMG distribution space as compared to fed cells (10 mM; 18 hours).(ABSTRACT TRUNCATED AT 400 WORDS)     

Cell motility: an interplay between local and non-local measurement

The bending motions of an ATP-activated actin filament and a flagellar axoneme of starfish spermatozoon exhibit a one-to-many correspondence between the displacement of the medium and the force actualized in the medium, demonstrating sharp contrast to the one-to-one correspondence in classical mechanics. Uniqueness of the actualized forces is lacking. Cell motility suggests the absence of a completely specifiable boundary condition that would unambiguously control the dynamics of generating mechanical forces in motile cells. The one-to-many relationship between the displacement of the medium and the force actualized in the medium necessitates a materialistic capacity for making choices on the part of molecules and cells in transforming future possibilities into actualized reality, the latter of which again serves as the source of the further future possibilities.     

Dynamic interplay between O-GlcNAc and O-phosphate: the sweet side of protein regulation

Beta-O-linked N-acetylglucosamine (O-GlcNAc) is an abundant modification of cytosolic and nuclear proteins that occurs in metazoans. O-GlcNAc is dynamically processed by a unique set of enzymes that actively add and remove the modification. Functionally, O-GlcNAc appears to regulate protein stability, subcellular localization and protein-protein interactions. The modification often acts in a reciprocal manner to O-phosphate modifications of proteins and together they can synergistically control the activity of many cellular processes. Recently, O-GlcNAc has been demonstrated to play a significant role in diseases such as diabetes, cancer and neurodegeneration. For example, the increased levels of O-GlcNAc that occur in diabetes are associated with decreased insulin responsiveness in adipocytes.     

Structure-function relationships in UCP1, UCP2 and chimeras: EPR analysis and retinoic acid activation of UCP2.

Uncoupling proteins (UCPs) are composed of three repeated domains of approximately 100 amino acids each. We have used chimeras of UCP1 and UCP2, and electron paramagnetic resonance (EPR), to investigate domain specific properties of these UCPs. Questions include: are the effects of nucleotide binding on proton transport solely mediated by amino acids in the third C-terminal domain, and are the amino acids in the first two domains involved in retinoic or fatty acid activation? We first confirmed that our reconstitution system produced UCP1 that exhibited known properties, such as activation by fatty acids and inhibition of proton transport by purine nucleotides. Our results confirm the observations reported for recombinant yeast that retinoic acid, but not fatty acids known to activate UCP1, activates proton transport by UCP2 and that this activation is insensitive to nucleotide inhibition. We constructed chimeras in which the last domains of UCP1 or UCP2 were switched and tested for activation by fatty acids or retinoic acid and inhibition by nucleotides. U1U2 is composed of mUCP1 (amino acids 1-198) and hUCP2 (amino acids 211-309). Fatty acids activated proton transport of U1U2 and GTP mediated inhibition. In the other chimeric construct U2U1, hUCP2 (amino acids 1-210) and mUCP1 (amino acids 199-307), retinoic acid still acted as an activator, but no inhibition was observed with GTP. Using EPR, a method well suited to the analysis of the structure of membrane proteins such as UCPs, we confirmed that UCP2 binds nucleotides. The EPR data show large structural changes in UCP1 and UCP2 on exposure to ATP, implying that a putative nucleotide-binding site is present on UCP2. EPR analysis also demonstrated changes in conformation of UCP1/UCP2 chimeras following exposure to purine nucleotides. These data demonstrate that a nucleotide-binding site is present in the C-terminal domain of UCP2. This domain was able to inhibit proton transport only when fused to the N-terminal part of UCP1 (chimera U1U2). Thus, residues involved in nucleotide inhibition of proton transport are located in the two first carrier motifs of UCP1. While these results are consistent with previously reported effects of the C-terminal domain on nucleotide binding, they also demonstrate that interactions with the N-terminal domains are necessary to inhibit proton transport. Finally, the results suggest that proteins such as UCP2 may transport protons even though they are not responsible for basal or cold-induced thermogenesis.