Molecular evolution of UCP1 and the evolutionary history of mammalian non-shivering thermogenesis

Background  

Uncoupling protein 1 (UCP1) is a mitochondrial anion carrier, expressed in brown adipose tissue (BAT) of Eutherians. UCP1 is responsible for uncoupling mitochondrial proton transport from the production of ATP, thereby dissipating heat; it is essential for non-shivering thermogenesis (NST) in mammalian BAT. UCP1 orthologs have been identified in non-Eutherian mammals, fish and amphibians. Yet, UCP1 has a unique function in Eutherians in that it is necessary in the production of heat (NST). As such, this study aims to determine the evolutionary mode of UCP1 in Eutherians, where there is clear evidence of UCP1-dependent NST in BAT.     

Metabolic Flux Analysis of Mitochondrial Uncoupling in 3T3-L1 Adipocytes

Background

Increasing energy expenditure at the cellular level offers an attractive option to limit adiposity and improve whole body energy balance. In vivo and in vitro observations have correlated mitochondrial uncoupling protein-1 (UCP1) expression with reduced white adipose tissue triglyceride (TG) content. The metabolic basis for this correlation remains unclear.

Methodology/Principal Findings

This study tested the hypothesis that mitochondrial uncoupling requires the cell to compensate for the decreased oxidation phosphorylation efficiency by up-regulating lactate production, thus redirecting carbon flux away from TG synthesis. Metabolic flux analysis was used to characterize the effects of non-lethal, long-term mitochondrial uncoupling (up to 18 days) on the pathways of intermediary metabolism in differentiating 3T3-L1 adipocytes. Uncoupling was induced by forced expression of UCP1 and chemical (FCCP) treatment. Chemical uncoupling significantly decreased TG content by ca. 35%. A reduction in the ATP level suggested diminished oxidative phosphorylation efficiency in the uncoupled adipocytes. Flux analysis estimated significant up-regulation of glycolysis and down-regulation of fatty acid synthesis, with chemical uncoupling exerting quantitatively larger effects.

Conclusions/Significance

The results of this study support our hypothesis regarding uncoupling-induced redirection of carbon flux into glycolysis and lactate production, and suggest mitochondrial proton translocation as a potential target for controlling adipocyte lipid metabolism.     

Cytochalasin B triggers a novel pertussis toxin sensitive pathway in TNF-alpha primed neutrophils

Background  

Cytochalasin B does not directly activate the oxygen-radical-producing NADPH oxidase activity of neutrophils but transfers desensitized G-protein coupled receptors (GPCR) into an active signaling state by uncoupling GCPR from the cytoskeleton. The receptor uncoupling results in respiratory burst activity when signals generated by reactivated formyl peptide receptors trigger the NADPH-oxidase to produce superoxide anions.     

Influence of CSP 310 and CSP 310-like proteins from cereals on mitochondrial energetic activity and lipid peroxidation in vitro and in vivo

Background  

The development of chilling and freezing injury symptoms in plants is known to frequently coincide with peroxidation of free fatty acids. Mitochondria are one of the major sources of reactive oxygen species during cold stress. Recently it has been suggested that uncoupling of oxidation and phosphorylation in mitochondria during oxidative stress can decrease ROS formation by mitochondrial respiratory chain generation. At the same time, it is known that plant uncoupling mitochondrial protein (PUMP) and other UCP-like proteins are not the only uncoupling system in plant mitochondria. All plants have cyanide-resistant oxidase (AOX) whose activation causes an uncoupling of respiration and oxidative phosphorylation. Recently it has been found that in cereals, cold stress protein CSP 310 exists, and that this causes uncoupling of oxidation and phosphorylation in mitochondria.     

‘Mild mitochondrial uncoupling’ induced protection against neuronal excitotoxicity requires AMPK activity

The preconditioning response conferred by a mild uncoupling of the mitochondrial membrane potential (Δψ_m) has been attributed to altered reactive oxygen species (ROS) production and mitochondrial Ca^2 + uptake within the cells. Here we have explored if altered cellular energetics in response to a mild mitochondrial uncoupling stimulus may also contribute to the protection. The addition of 100 nM FCCP for 30 min to cerebellar granule neurons (CGNs) induced a transient depolarization of the Δψ_m, that was sufficient to significantly reduce CGN vulnerability to the excitotoxic stimulus, glutamate. On investigation, the mild mitochondrial ‘uncoupling’ stimulus resulted in a significant increase in the plasma membrane levels of the glucose transporter isoform 3, with a hyperpolarisation of Δψ_m and increased cellular ATP levels also evident following the washout of FCCP. Furthermore, the phosphorylation state of AMP-activated protein kinase (AMPK) (Thr 172) was increased within 5 min of the uncoupling stimulus and elevated up to 1 h after washout. Significantly, the physiological changes and protection evident after the mild uncoupling stimulus were lost in CGNs when AMPK activity was inhibited. This study identifies an additional mechanism through which protection is mediated upon mild mitochondrial uncoupling: it implicates increased AMPK signalling and an adaptive shift in energy metabolism as mediators of the preconditioning response associated with FCCP-induced mild mitochondrial uncoupling.     

Muscle Mitochondrial Uncoupling Dismantles Neuromuscular Junction and Triggers Distal Degeneration of Motor Neurons

Background

Amyotrophic lateral sclerosis (ALS), the most frequent adult onset motor neuron disease, is associated with hypermetabolism linked to defects in muscle mitochondrial energy metabolism such as ATP depletion and increased oxygen consumption. It remains unknown whether muscle abnormalities in energy metabolism are causally involved in the destruction of neuromuscular junction (NMJ) and subsequent motor neuron degeneration during ALS.

Methodology/Principal Findings

We studied transgenic mice with muscular overexpression of uncoupling protein 1 (UCP1), a potent mitochondrial uncoupler, as a model of muscle restricted hypermetabolism. These animals displayed age-dependent deterioration of the NMJ that correlated with progressive signs of denervation and a mild late-onset motor neuron pathology. NMJ regeneration and functional recovery were profoundly delayed following injury of the sciatic nerve and muscle mitochondrial uncoupling exacerbated the pathology of an ALS animal model.

Conclusions/Significance

These findings provide the proof of principle that a muscle restricted mitochondrial defect is sufficient to generate motor neuron degeneration and suggest that therapeutic strategies targeted at muscle metabolism might prove useful for motor neuron diseases.     

Up-regulation of avian uncoupling protein in cold-acclimated and hyperthyroid ducklings prevents reactive oxygen species production by skeletal muscle mitochondria

Background  

Although identified in several bird species, the biological role of the avian homolog of mammalian uncoupling proteins (avUCP) remains extensively debated. In the present study, the functional properties of isolated mitochondria were examined in physiological or pharmacological situations that induce large changes in avUCP expression in duckling skeletal muscle.     

Roles of the creatine kinase system and myoglobin in maintaining energetic state in the working heart

Background  

The heart is capable of maintaining contractile function despite a transient decrease in blood flow and increase in cardiac ATP demand during systole. This study analyzes a previously developed model of cardiac energetics and oxygen transport to understand the roles of the creatine kinase system and myoglobin in maintaining the ATP hydrolysis potential during beat-to-beat transient changes in blood flow and ATP hydrolysis rate.     

Identification of ATP binding residues of a protein from its primary sequence

Background  

One of the major challenges in post-genomic era is to provide functional annotations for large number of proteins arising from genome sequencing projects. The function of many proteins depends on their interaction with small molecules or ligands. ATP is one such important ligand that plays critical role as a coenzyme in the functionality of many proteins. There is a need to develop method for identifying ATP interacting residues in a ATP binding proteins (ABPs), in order to understand mechanism of protein-ligands interaction.     

Reversible mitochondrial uncoupling in the cold phase during liver preservation/reperfusion reduces oxidative injury in the rat model

Reversible uncoupling of the mitochondrial electron-transport chain may be one strategy to prevent intracellular oxidative stress during liver cold preservation/warm reperfusion (CP/WR) injury. 2,4-Dinitrophenol (DNP) is a potent water-soluble uncoupling agent for supplementation of the hepatic CP solution. The aim of this work was to investigate the possible influence of DNP in the CP solution on the isolated rat liver state during CP/WR. Livers were subjected to CP at 4 °C in sucrose–phosphate based solution (SPS) for 18 h, followed by WR for 60 min in vitro. The final concentration of DNP was 100 μM. DNP presence during the CP stage led to partial ATP level decrease accompanied by a significant diminution in liver TBARS and a prevention of antioxidant enzyme activity decrease (catalase, GSH-PO, GSH-Red) when compared with livers stored without DNP. After DNP wash-out during WR, an improvement in the mitochondrial functional state (higher respiratory control indices and ATP levels, and a decrease in V₄ respiration rates) were observed. This was concurrent with lower TBARS levels, higher antioxidant enzyme activities and significant increase of bile production. The results suggest that reversible uncoupling may be one way to influence oxidative stress associated with hepatic cold preservation.     

Bridging the phenotypic gap: Real-time assessment of mitochondrial function and metabolism of the nematode <Emphasis Type="Italic">Caenorhabditis elegans</Emphasis>

Background  

The ATP levels of an organism are an important physiological parameter that is affected by genetic make up, ageing, stress and disease.     

γ-Irradiation induces P2X7 receptor-dependent ATP release from B16 melanoma cells

Background

Ionizing irradiation causes not only growth arrest and cell death, but also release of growth factors or signal transmitters, which promote cancer malignancy. Extracellular ATP controls cancer growth through activation of purinoceptors. However, there is no report of radiation-induced ATP release from cancer cells. Here, we examined γ-irradiation-induced ATP release and its mechanism in B16 melanoma.

Methods

Extracellular ATP was measured by luciferin–luciferase assay. To investigate mechanism of radiation-induced ATP release, we pharmacologically inhibited the ATP release and established stable P2X₇ receptor-knockdown B16 melanoma cells using two short hairpin RNAs targeting P2X₇ receptor.

Results

Cells were exposed to 0.5–8 Gy of γ-rays. Extracellular ATP was increased, peaking at 5 min after 0.5 Gy irradiation. A selective P2X₇ receptor channel antagonist, but not anion transporter inhibitors, blocked the release of ATP. Further, radiation-induced ATP release was significantly decreased in P2X₇ receptor-knockdown cells. Our results indicate that γ-irradiation evokes ATP release from melanoma cells, and P2X₇ receptor channel plays a significant role in mediating the ATP release.

General Significance

We suggest that extracellular ATP could be a novel intercellular signaling molecule released from cancer cells when cells are exposed to ionizing radiation.     

Selective Protection of Human Liver Tissue in TNF-Targeting of Cancers of the Liver by Transient Depletion of Adenosine Triphosphate

Background

Tumor necrosis factor alpha (TNF) is able to kill cancer cells via receptor-mediated cell death requiring adenosine triphosphate (ATP). Clinical usage of TNF so far is largely limited by its profound hepatotoxicity. Recently, it was found in the murine system that specific protection of hepatocytes against TNF''s detrimental effects can be achieved by fructose-mediated ATP depletion therein. Before employing this quite attractive selection principle in a first clinical trial, we here comprehensively investigated the interdependence between ATP depletion and TNF hepatotoxicity in both in vitro and ex vivo experiments based on usage of primary patient tissue materials.

Methods

Primary human hepatocytes, and both non-tumorous and tumorous patient-derived primary liver tissue slices were used to elucidate fructose-induced ATP depletion and TNF-induced cytotoxicity.

Results

PHH as well as tissue slices prepared from non-malignant human liver specimen undergoing a fructose-mediated ATP depletion were both demonstrated to be protected against TNF-induced cell death. In contrast, due to tumor-specific overexpression of hexokinase II, which imposes a profound bypass on hepatocytic-specific fructose catabolism, this was not the case for human tumorous liver tissues.

Conclusion

Normal human liver tissues can be protected transiently against TNF-induced cell death by systemic pretreatment with fructose used in non-toxic/physiologic concentrations. Selective TNF-targeting of primary and secondary tumors of the liver by transient and specific depletion of hepatocytic ATP opens up a new clinical avenue for the TNF-based treatment of liver cancers.     

Transfection of oral squamous cell carcinoma with human papillomavirus-16 induces proliferative and morphological changes in vitro

Background  

Human papillomavirus has been implicated in virtually all cervical cancers and is believed to be the primary etiological factor that transforms cervical epithelia. The presence of HPV in oral cancers suggests that HPV may play a similar role in transforming the oral epithelia. The prevalence of HPV in oral cancers is highly variable, however, presenting problematic issues regarding the etiology of oral cancers, which must be investigated more thoroughly. Past analyses of HPV in cancers of the oral cavity have largely been confined to retrospective studies of cancer patients. The purpose of this study was to examine the potential for HPV16 infection to alter the proliferative phenotype of oral squamous cell carcinoma in vitro.     

Combination of apigenin treatment with therapeutic HPV DNA vaccination generates enhanced therapeutic antitumor effects

Background  

It is important to develop innovative therapies for advanced stage cancers in addition to the conventional therapies including chemotherapy, radiation and surgery. Antigen-specific immunotherapy has emerged as a novel alternate therapy for advanced stage cancers, which may be employed in conjunction with conventional therapies.     

Plasma ATP concentration and venous oxygen content in the forearm during dynamic handgrip exercise

Background  

It has been proposed that adenosine triphosphate (ATP) released from red blood cells (RBCs) may contribute to the tight coupling between blood flow and oxygen demand in contracting skeletal muscle. To determine whether ATP may contribute to the vasodilatory response to exercise in the forearm, we measured arterialised and venous plasma ATP concentration and venous oxygen content in 10 healthy young males at rest, and at 30 and 180 seconds during dynamic handgrip exercise at 45% of maximum voluntary contraction (MVC).     

Identification and validation of genes involved in gastric tumorigenesis

Background  

Gastric cancer is one of the common cancers seen in south India. Unfortunately more than 90% are advanced by the time they report to a tertiary centre in the country. There is an urgent need to characterize these cancers and try to identify potential biomarkers and novel therapeutic targets.     

PPAR-γ Regulates Carnitine Homeostasis and Mitochondrial Function in a Lamb Model of Increased Pulmonary Blood Flow

Objective

Carnitine homeostasis is disrupted in lambs with endothelial dysfunction secondary to increased pulmonary blood flow (Shunt). Our recent studies have also indicated that the disruption in carnitine homeostasis correlates with a decrease in PPAR-γ expression in Shunt lambs. Thus, this study was carried out to determine if there is a causal link between loss of PPAR-γ signaling and carnitine dysfunction, and whether the PPAR-γ agonist, rosiglitazone preserves carnitine homeostasis in Shunt lambs.

Methods and Results

siRNA-mediated PPAR-γ knockdown significantly reduced carnitine palmitoyltransferases 1 and 2 (CPT1 and 2) and carnitine acetyltransferase (CrAT) protein levels. This decrease in carnitine regulatory proteins resulted in a disruption in carnitine homeostasis and induced mitochondrial dysfunction, as determined by a reduction in cellular ATP levels. In turn, the decrease in cellular ATP attenuated NO signaling through a reduction in eNOS/Hsp90 interactions and enhanced eNOS uncoupling. In vivo, rosiglitazone treatment preserved carnitine homeostasis and attenuated the development of mitochondrial dysfunction in Shunt lambs maintaining ATP levels. This in turn preserved eNOS/Hsp90 interactions and NO signaling.

Conclusion

Our study indicates that PPAR-γ signaling plays an important role in maintaining mitochondrial function through the regulation of carnitine homeostasis both in vitro and in vivo. Further, it identifies a new mechanism by which PPAR-γ regulates NO signaling through Hsp90. Thus, PPAR-γ agonists may have therapeutic potential in preventing the endothelial dysfunction in children with increased pulmonary blood flow.     

Cell-free protein synthesis energized by slowly-metabolized maltodextrin

Background  

Cell-free protein synthesis (CFPS) is a rapid and high throughput technology for obtaining proteins from their genes. The primary energy source ATP is regenerated from the secondary energy source through substrate phosphorylation in CFPS.     

Both ischemic preconditioning and ghrelin administration protect hippocampus from ischemia/reperfusion and upregulate uncoupling protein-2

Background  

A major endogenous protective mechanism in many organs against ischemia/reperfusion (I/R) injury is ischemic preconditioning (IPC). By moderately uncoupling the mitochondrial respiratory chain and decreasing production of reactive oxygen species (ROS), IPC reduces apoptosis induced by I/R by reducing cytochrome c release from the mitochondria. One element believed to contribute to reduce ROS production is the uncoupling protein UCP2 (and UCP3 in the heart). Although its implication in IPC in the brain has been shown in vitro, no in vivo study of protein has shown its upregulation. Our first goal was to determine in rat hippocampus whether UCP2 protein upregulation was associated with IPC-induced protection and increased ROS production. The second goal was to determine whether the peptide ghrelin, which possesses anti-oxidant and protective properties, alters UCP2 mRNA levels in the same way as IPC during protection.