Cumulative insults to mitochondrial function may promote the emergence of 'syndrome X' and diabetes mellitus in HIV/HCV co-infected patients

The 'metabolic syndrome' or 'syndrome X' affects approximately a quarter of the general population of the United States. A significant number of patients, therefore, may have predisposing risk factors for developing 'syndrome X' and/or diabetes mellitus (DM) prior to HIV infection, hepatitis C (HCV) infection, and/or institution of highly active antiretroviral therapy (HAART). Metabolic perturbations identical to 'syndrome X' develop in many HIV-infected patients who are undergoing HAART. A series of cumulative and possibly synergistic insults to mitochondrial function which occur in HIV/HCV co-infected patients who are receiving HAART may promote the emergence of 'syndrome X' and DM. HIV-infected patients, especially those who are co-infected with HCV and who are undergoing HAART, may, therefore, be at increased risk of developing the cardiovascular complications that are associated with 'syndrome X' and DM.     

Increased uncoupling protein-2 and -3 gene expressions in skeletal muscle of STZ-induced diabetic rats

Streptozotocin (STZ)-induced diabetic animals are vulnerable to cold stress. Uncoupling proteins (UCPs) play an important role in regulating thermogenesis. We investigated the gene expressions of UCPs in brown adipose tissue (BAT), white adipose tissue (WAT), liver and gastrocnemius muscle of STZ-diabetic rats using Northern blot. UCP-1, -2 and -3 mRNA expressions in BAT were all remarkably lower in STZ-diabetic rats than those in control rats. Both UCP-2 and -3 gene expressions in gastrocnemius muscle were substantially elevated in STZ-diabetic rats and insulin treatment restored UCP gene expressions to normal levels. These results suggest that in STZ-diabetic rats, the overexpression of UCP-2 and UCP-3 in skeletal muscle provides a defense against hypothermogenesis caused by decreased UCPs in BAT.     

Mitochondrial uncoupling proteins in unicellular eukaryotes

Uncoupling proteins (UCPs) are members of the mitochondrial anion carrier protein family that are present in the mitochondrial inner membrane and mediate free fatty acid (FFA)-activated, purine nucleotide (PN)-inhibited proton conductance. Since 1999, the presence of UCPs has been demonstrated in some non-photosynthesising unicellular eukaryotes, including amoeboid and parasite protists, as well as in non-fermentative yeast and filamentous fungi. In the mitochondria of these organisms, UCP activity is revealed upon FFA-induced, PN-inhibited stimulation of resting respiration and a decrease in membrane potential, which are accompanied by a decrease in membranous ubiquinone (Q) reduction level. UCPs in unicellular eukaryotes are able to divert energy from oxidative phosphorylation and thus compete for a proton electrochemical gradient with ATP synthase. Our recent work indicates that membranous Q is a metabolic sensor that might utilise its redox state to release the PN inhibition of UCP-mediated mitochondrial uncoupling under conditions of phosphorylation and resting respiration. The action of reduced Q (QH₂) could allow higher or complete activation of UCP. As this regulatory feature was demonstrated for microorganism UCPs (A. castellanii UCP), plant and mammalian UCP1 analogues, and UCP1 in brown adipose tissue, the process could involve all UCPs. Here, we discuss the functional connection and physiological role of UCP and alternative oxidase, two main energy-dissipating systems in the plant-type mitochondrial respiratory chain of unicellular eukaryotes, including the control of cellular energy balance as well as preventive action against the production of reactive oxygen species.     

G protein-coupled receptors for free fatty acids

Free fatty acids (FFAs) are not only an important direct source of energy but they also play key roles in regulating a range of physiological responses. Although it was long assumed that such effects of FFAs must occur following cellular uptake, and potentially via their conversion to fatty acyl-CoAs, it is now apparent that FFAs also function directly as agonists at a number of G protein-coupled receptors (GPCRs). Tissue distribution studies and, subsequently, siRNA knock-down experiments have indicated key roles for these GPCRs in glucose homeostasis, adipogenesis, white cell recruitment and potentially in a range of other processes. Considerable interest is thus now centred on the generation of potent and selective small molecule ligands, both as tool compounds to further unravel the biology and physiological role of this group of GPCRs and as starting points for possible therapeutic intervention in a range of areas, particularly those associated with 'metabolic syndrome'.     

UCP2, UCP3, avUCP, what do they do when proton transport is not stimulated? Possible relevance to pyruvate and glutamine metabolism

Uncoupling proteins (UCPs) are specialized members of the mitochondrial transporter family. They allow passive proton transport through the mitochondrial inner membrane. This activity leads to uncoupling of mitochondrial respiration and to energy waste, which is well documented with UCP1 in brown adipose tissue. The uncoupling activity of the new UCPs (discovered after 1997), such as UCP2 and UCP3 in mammals or avUCP in birds, is more difficult to characterize. However, extensive data support the idea that the new UCPs are involved in the control of reactive oxygen species (ROS) generation. This fits with the hypothesis that mild uncoupling caused by the UCPs prevents ROS production. Activators and inhibitors regulate the proton transport activity of the UCPs. In the absence of activators of proton transport, the UCP allows the permeation of other ions. We suggest that this activity has physiological significance and, for example, UCP3 expressed in glycolytic muscle fibres may be a passive pyruvate transporter ensuring equilibrium between glycolysis and oxidative phosphorylation. Induction of UCP2 expression by glutamine strengthens the proposal that new UCPs could act to determine the choice of mitochondrial substrate. This would obviously have an impact on mitochondrial bioenergetics and ROS production.     

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.     

Fasting increases gene expressions of uncoupling proteins and peroxisome proliferator-activated receptor-gamma in brown adipose tissue of ventromedial hypothalamus-lesioned rats

Uncoupling proteins (UCPs) are supposed to be involved in diet-induced thermogenesis. Their activities are usually elevated by feeding and reduced by fasting in normal animals. To investigate whether fasting affects the expression of UCPs mRNA in brown adipose tissue (BAT) of bilateral ventromedial hypothalamus (VMH)-lesioned rats, we determined the gene expression of UCP1, UCP2 or UCP3 in BAT of VMH-lesioned rats and examined oxygen consumption in these rats under fed or 48-h fasted conditions. Northern blotting revealed no difference in the expression of UCPs mRNA in BAT between VMH-lesioned and sham-operated rats under the fed condition, however, expressions were increased markedly in BAT of VMH-lesioned rats under the fasted condition. Under the fed condition, no difference in oxygen consumption was observed between VMH-lesioned and sham-operated rats. Under the fasted condition, oxygen consumption decreased in both rats, however, it decreased in VMH-lesioned less than in sham operated rats. To explore the mechanism that fasting elevated BAT UCPs mRNA in VMH-lesioned rats, we measured peroxisome proliferator-activated receptor (PPAR)-gamma mRNA and protein in BAT, because PPAR-gamma agonist can elevate UCPs mRNA levels in BAT. Under the fed condition, no differences in the expression of PPAR-gamma mRNA and protein content were observed between in BAT of VMH-lesioned and sham-operated rats. Under the fasted condition, however, both increased in BAT of VMH-lesioned rats. These results suggest that VMH-lesions enhance the gene expression of UCPs in BAT under long-term fasting as a defensive reaction to inhibit the reduction of body temperature through an increase in PPAR-gamma activity.     

Further support to the uncoupling-to-survive theory: the genetic variation of human UCP genes is associated with longevity

In humans Uncoupling Proteins (UCPs) are a group of five mitochondrial inner membrane transporters with variable tissue expression, which seem to function as regulators of energy homeostasis and antioxidants. In particular, these proteins uncouple respiration from ATP production, allowing stored energy to be released as heat. Data from experimental models have previously suggested that UCPs may play an important role on aging rate and lifespan. We analyzed the genetic variability of human UCPs in cohorts of subjects ranging between 64 and 105 years of age (for a total of 598 subjects), to determine whether specific UCP variability affects human longevity. Indeed, we found that the genetic variability of UCP2, UCP3 and UCP4 do affect the individual's chances of surviving up to a very old age. This confirms the importance of energy storage, energy use and modulation of ROS production in the aging process. In addition, given the different localization of these UCPs (UCP2 is expressed in various tissues including brain, hearth and adipose tissue, while UCP3 is expressed in muscles and Brown Adipose Tissue and UCP4 is expressed in neuronal cells), our results may suggest that the uncoupling process plays an important role in modulating aging especially in muscular and nervous tissues, which are indeed very responsive to metabolic alterations and are very important in estimating health status and survival in the elderly.     

The conserved regulation of mitochondrial uncoupling proteins: From unicellular eukaryotes to mammals

Uncoupling proteins (UCPs) belong to the mitochondrial anion carrier protein family and mediate regulated proton leak across the inner mitochondrial membrane. Free fatty acids, aldehydes such as hydroxynonenal, and retinoids activate UCPs. However, there are some controversies about the effective action of retinoids and aldehydes alone; thus, only free fatty acids are commonly accepted positive effectors of UCPs. Purine nucleotides such as GTP inhibit UCP-mediated mitochondrial proton leak. In turn, membranous coenzyme Q may play a role as a redox state-dependent metabolic sensor that modulates the complete activation/inhibition of UCPs. Such regulation has been observed for UCPs in microorganisms, plant and animal UCP1 homologues, and UCP1 in mammalian brown adipose tissue. The origin of UCPs is still under debate, but UCP homologues have been identified in all systematic groups of eukaryotes. Despite the differing levels of amino acid/DNA sequence similarities, functional studies in unicellular and multicellular organisms, from amoebae to mammals, suggest that the mechanistic regulation of UCP activity is evolutionarily well conserved. This review focuses on the regulatory feedback loops of UCPs involving free fatty acids, aldehydes, retinoids, purine nucleotides, and coenzyme Q (particularly its reduction level), which may derive from the early stages of evolution as UCP first emerged.     

Long-term challenges in growth hormone treatment

Growth hormone deficiency (GHD) is defined biochemically as a response to hypoglycaemia with a peak GH concentration of less than 5 microg/l. The 'GHD syndrome' is a range of psychological and physical symptoms that are associated with GHD, which include increased central adiposity, decreased bone mineral density, abnormal lipid profiles, decreased cardiovascular performance, reduced lean body mass (LBM), social isolation, depressed mood and increased anxiety. Importantly, the combination of physical and psychological problems can often result in a reduced quality of life. A number of trials have shown that GH replacement therapy can lead to a substantial improvement in GHD associated symptoms. Following up to 12 months of treatment with GH, LBM increased, left ventricular systolic function improved and the mean volume of adipose tissue fell. After only 4 months of treatment, a rise in exercise capacity was recorded, and after 2 years' treatment, isokinetic and isometric muscle strength had normalized in proximal muscle groups. Feelings of well-being and vitality also improved significantly. However, studies on the effects of treatment on insulin sensitivity in GH-deficient patients have had conflicting results. In this paper, we will discuss the long-term consequences of GHD and the effects of GH replacement therapy.     

禁食及胰岛素水平对大鼠解偶联蛋白-1、2、3基因表达的影响

 为探讨禁食和胰岛素对解偶联蛋白 - 1、2、3基因 (UCP1 ,2 ,3)表达的影响 ,应用 RT- PCR方法观察了在不同禁食时间和应用胰岛素条件下大鼠白色脂肪组织、棕色脂肪组织和骨骼肌中 UCP1 ,2 ,3m RNA水平的变化 .UCP1基因只在大鼠棕色脂肪组织中表达 .UCP2 ,3基因在三种组织中均有表达 ,在白色脂肪组织中以 UCP2表达为主 ;在骨骼肌中以 UCP3表达为主 .过夜禁食使棕色脂肪组织 UCP1 ,3m RNA水平明显下降 (P<0 .0 1 ) ;UCP2 m RNA水平在三种组织中均呈上升反应 ,以白色脂肪组织中表现最为明显 (P<0 .0 5) ;而对白色脂肪组织和骨骼肌中 UCP3基因表达无明显影响 .禁食时间延长至 48h,除棕色脂肪组织中 UCP2 ,3基因有明显下降外 ,各组织中UCPs基因表达基本调节至正常或高于对照组水平 .胰岛素对 UCPs基因表达水平有一定的上调作用 ,这一作用对棕色脂肪组织 UCPs各基因及骨骼肌中 UCP3基因表现得尤为明显 (P<0 .0 5) .大鼠 UCPs基因表达有一定的组织特异性 ;禁食时间对三种组织中 UCPs各成员基因表达的影响有时相上的区别 ;胰岛素可以调 UCPs各成员基因的表达 .结果反映了 UCPs各成员在能量代谢调节上的不同作用 ,这为理解膳食 -产热与体重调节的关系 ,及其能量代谢平衡与疾病关系提供了实验依据     

Uncoupling proteins and thermoregulation.

Energy balance in animals is a metabolic state that exists when total body energy expenditure equals dietary energy intake. Energy expenditure, or thermogenesis, can be subcategorized into groups of obligatory and facultative metabolic processes. Brown adipose tissue (BAT), through the activity of uncoupling protein 1 (UCP1), is responsible for nonshivering thermogenesis, a major component of facultative thermogenesis in newborn humans and in small mammals. UCP1, found in the mitochondrial inner membrane in BAT, uncouples energy substrate oxidation from mitochondrial ATP production and hence results in the loss of potential energy as heat. Mice that do not express UCP1 (UCP1 knockouts) are markedly cold sensitive. The recent identification of four new homologs to UCP1 expressed in BAT, muscle, white adipose tissue, brain, and other tissues has been met by tremendous scientific interest. The hypothesis that the novel UCPs may regulate thermogenesis and/or fatty acid metabolism guides investigations worldwide. Despite several hundred publications on the new UCPs, there are a number of significant controversies, and only a limited understanding of their physiological and biochemical properties has emerged. The discovery of UCP orthologs in fish, birds, insects, and even plants suggests the widespread importance of their metabolic functions. Answers to fundamental questions regarding the metabolic functions of the new UCPs are thus pending and more research is needed to elucidate their physiological functions. In this review, we discuss recent findings from mammalian studies in an effort to identify potential patterns of function for the UCPs.     

Upregulation of uncoupling protein homologues in skeletal muscle but not adipose tissue in posttraumatic insulin resistance

Metabolic alterations after surgical stress include peripheral insulin resistance and increased utilization of fat as a fuel substrate. An up-regulation of skeletal muscle uncoupling proteins (UCPs) has been associated with physiologic states of insulin resistance and enhanced fat metabolism in rodents. We examined whether posttraumatic insulin resistance induced the UCPs in gastrocnemius and soleus muscle and white adipose tissue in an experimental model of surgical trauma. Insulin sensitivity was significantly reduced in isolated soleus muscles but unchanged in adipocytes after trauma. In traumatized rats, mRNA and protein contents of UCP2 and UCP3 and were significantly increased in both muscle types. UCP2 protein content in adipose tissue was unaltered by surgical stress. Circulating NEFAs and glycerol were reduced after surgical trauma. We hypothesize that the changes in UCP2 and UCP3 gene and protein expression are involved in the regulation of substrate utilization in posttraumatic insulin resistance.     

Mitochondrial uncoupling proteins: regulation and physiological role

Uncoupling proteins (UCPs), members of mitochondrial carrier family, are present in mitochondrial inner membrane and mediate free fatty acid-activated, purine-nucleotide-inhibited H+ re-uptake. UCPs can modulate the tightness of coupling between mitochondrial respiration and ATP synthesis. A physiological function of the first described UCP, UCP1 or termogenin, present in mitochondria of mammalian brown adipose tissues is well established. UCP1 plays a role in nonshivering thermogenesis in mammals. The widespread presence of UCPs in eukaryotes, in non-thermogenic tissues of animals, plants and in unicellular organisms implies that these proteins may elicit other functions than thermogenesis. However, the physiological functions of UCP1 homologues are still under debate. They can regulate energy metabolism through modulation of the electrochemical proton gradient and production of ROS. Functional activation of UCPs is proposed to decrease ROS production. Moreover, products of lipid peroxidation can activate UCPs and promote feedback down-regulation of mitochondrial ROS production.     

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.     

Role of fatty acids in the transition from anaerobic to aerobic metabolism in skeletal muscle during exercise

In moderate physical exercise, the transition from predominantly anaerobic towards predominantly aerobic metabolism is a key step to improve performance. Increase in the supply of oxygen and nutrients, such as free fatty acids (FFA) and glucose, which accompanies high blood flow, is required for this transition. The mechanisms involved in the vasodilation in skeletal muscle during physical activity are not completely known yet. In this article, we postulate a role of FFA and heat production in this process. The presence of uncoupling protein-2 and -3 (UCP-2 and -3) in skeletal muscle, whose activity is dependent on FFA, suggests that these metabolites can act as mitochondrial uncouplers in this tissue. Evidence indicates however that UCPs act as uncouplers only when coenzyme Q is predominantly in the reduced state (i.e. under nonphosphorylation conditions or state 4 respiration) as is observed in resting muscles and in the beginning of physical activity (predominantly anaerobic metabolism). The increase in the lipolytic activity in adipose tissue in the beginning of physical activity results in elevated plasma FFA levels. The FFA can then act on the UCPs, increasing the local heat production. We propose that this calorigenic effect of FFA is important to activate nitric oxide synthase, resulting in nitric oxide production and consequent vasodilation. Therefore, FFA would be important mediators for the changes that occur in muscle metabolism during prolonged physical activity, ensuring the appropriate supply of oxygen and nutrients by increasing blood flow at the beginning of exercise in the contracting skeletal muscles.     

Cell-free production and characterisation of human uncoupling protein 1–3

The uncoupling proteins (UCPs) leak protons across the inner mitochondrial membrane, thus uncoupling the proton gradient from ATP synthesis. The main known physiological role for this is heat generation by UCP1 in brown adipose tissue. However, UCPs are also believed to be important for protection against reactive oxygen species, fine-tuning of metabolism and have been suggested to be involved in disease states such as obesity, diabetes and cancer.Structural studies of UCPs have long been hampered by difficulties in sample preparation with neither expression in yeast nor refolding from inclusion bodies in E. coli yielding sufficient amounts of pure and stable protein. In this study, we have developed a protocol for cell-free expression of human UCP1, 2 and 3, resulting in 1 mg pure protein per 20 mL of expression media. Lauric acid, a natural UCP ligand, significantly improved protein thermal stability and was therefore added during purification. Secondary structure characterisation using circular dichroism spectroscopy revealed the proteins to consist of mostly α-helices, as expected. All three UCPs were able to bind GDP, a well-known physiological inhibitor, as shown by the Fluorescence Resonance Energy Transfer (FRET) technique, suggesting that the proteins are in a natively folded state.     

Functional characterization of UCP1 in mammalian HEK293 cells excludes mitochondrial uncoupling artefacts and reveals no contribution to basal proton leak

Mechanistic studies on uncoupling proteins (UCPs) not only are important to identify their cellular function but also are pivotal to identify potential drug targets to manipulate mitochondrial energy transduction. So far, functional and comparative studies of uncoupling proteins in their native environment are hampered by different mitochondrial, cellular and genetic backgrounds. Artificial systems such as yeast ectopically expressing UCPs or liposomes with reconstituted UCPs were employed to address crucial mechanistic questions but these systems also produced inconsistencies with results from native mitochondria. We here introduce a novel mammalian cell culture system (Human Embryonic Kidney 293 - HEK293) to study UCP1 function. Stably transfected HEK293 cell lines were derived that contain mouse UCP1 at concentrations comparable to tissue mitochondria. In this cell-based test system UCP1 displays native functional behaviour as it can be activated with fatty acids (palmitate) and inhibited with purine nucleotides guanosine-diphosphate (GDP). The catalytic centre activity of the UCP1 homodimer in HEK293 is comparable to activities in brown adipose tissue supporting functionality of UCP1. Importantly, at higher protein levels than in yeast mitochondria, UCP1 in HEK293 cell mitochondria is fully inhibitable and does not contribute to basal proton conductance, thereby emphasizing the requirement of UCP1 activation for therapeutic purposes. These findings and resulting analysis on UCP1 characteristics demonstrate that the mammalian HEK293 cell system is suitable for mechanistic and comparative functional studies on UCPs and provides a non-confounding mitochondrial, cellular and genetic background.