Early detection of skeletal muscle bioenergetic deficit by magnetic resonance spectroscopy in cigarette smoke-exposed mice.

Skeletal muscle dysfunction is a common complication and an important prognostic factor in patients with chronic obstructive pulmonary disease (COPD). It is associated with intrinsic muscular abnormalities of the lower extremities, but it is not known whether there is an easy way to predict its presence. Using a mouse model of chronic cigarette smoke exposure, we tested the hypothesis that magnetic resonance spectroscopy allows us to detect muscle bioenergetic deficit in early stages of lung disease. We employed this technique to evaluate the synthesis rate of adenosine triphosphate (ATP) and characterize concomitant mitochondrial dynamics patterns in the gastrocnemius muscle of emphysematous mice. The fibers type composition and citrate synthase (CtS) and cytochrome c oxidase subunit IV (COX4) enzymatic activities were evaluated. We found that the rate of ATP synthesis was reduced in the distal skeletal muscle of mice exposed to cigarette smoke. Emphysematous mice showed a significant reduction in body weight gain, in the cross-sectional area of the total fiber and in the COX4 to CtS activity ratio, due to a significant increase in CtS activity of the gastrocnemius muscle. Taken together, these data support the hypothesis that in the early stage of lung disease, we can detect a decrease in ATP synthesis in skeletal muscle, partly caused by high oxidative mitochondrial enzyme activity. These findings may be relevant to predict the presence of skeletal bioenergetic deficit in the early stage of lung disease besides placing the mitochondria as a potential therapeutic target for the treatment of COPD comorbidities.

Evaluation of energy metabolism and calcium homeostasis in cells affected by Shwachman-Diamond syndrome.

Isomorphic mutation of the SBDS gene causes Shwachman-Diamond syndrome (SDS). SDS is a rare genetic bone marrow failure and cancer predisposition syndrome. SDS cells have ribosome biogenesis and their protein synthesis altered, which are two high-energy consuming cellular processes. The reported changes in reactive oxygen species production, endoplasmic reticulum stress response and reduced mitochondrial functionality suggest an energy production defect in SDS cells. In our work, we have demonstrated that SDS cells display a Complex IV activity impairment, which causes an oxidative phosphorylation metabolism defect, with a consequent decrease in ATP production. These data were confirmed by an increased glycolytic rate, which compensated for the energetic stress. Moreover, the signalling pathways involved in glycolysis activation also appeared more activated; i.e. we reported AMP-activated protein kinase hyper-phosphorylation. Notably, we also observed an increase in a mammalian target of rapamycin phosphorylation and high intracellular calcium concentration levels ([Ca(2+)]i), which probably represent new biochemical equilibrium modulation in SDS cells. Finally, the SDS cell response to leucine (Leu) was investigated, suggesting its possible use as a therapeutic adjuvant to be tested in clinical trials.

Coenzyme Q biosynthesis in health and disease.

Coenzyme Q (CoQ, or ubiquinone) is a remarkable lipid that plays an essential role in mitochondria as an electron shuttle between complexes I and II of the respiratory chain, and complex III. It is also a cofactor of other dehydrogenases, a modulator of the permeability transition pore and an essential antioxidant. CoQ is synthesized in mitochondria by a set of at least 12 proteins that form a multiprotein complex. The exact composition of this complex is still unclear. Most of the genes involved in CoQ biosynthesis (COQ genes) have been studied in yeast and have mammalian orthologues. Some of them encode enzymes involved in the modification of the quinone ring of CoQ, but for others the precise function is unknown. Two genes appear to have a regulatory role: COQ8 (and its human counterparts ADCK3 and ADCK4) encodes a putative kinase, while PTC7 encodes a phosphatase required for the activation of Coq7. Mutations in human COQ genes cause primary CoQ(10) deficiency, a clinically heterogeneous mitochondrial disorder with onset from birth to the seventh decade, and with clinical manifestation ranging from fatal multisystem disorders, to isolated encephalopathy or nephropathy. The pathogenesis of CoQ(10) deficiency involves deficient ATP production and excessive ROS formation, but possibly other aspects of CoQ(10) function are implicated. CoQ(10) deficiency is unique among mitochondrial disorders since an effective treatment is available. Many patients respond to oral CoQ(10) supplementation. Nevertheless, treatment is still problematic because of the low bioavailability of the compound, and novel pharmacological approaches are currently being investigated. This article is part of a Special Issue entitled 'EBEC 2016: 19th European Bioenergetics Conference, Riva del Garda, Italy, July 2-6, 2016', edited by Prof. Paolo Bernardi.

Dehydroabietic acid derivative QC2 induces oncosis in hepatocellular carcinoma cells.

AIM: Rosin, the traditional Chinese medicine, is reported to be able to inhibit skin cancer cell lines. In this report, we investigate the inhibitory effect against HCC cells of QC2, the derivative of rosin's main components dehydroabietic acid. METHODS: MTT assay was used to determine the cytotoxicity of QC2. Morphological changes were observed by time-lapse microscopy and transmission electron microscopy and the cytoskeleton changes were observed by laser-scanning confocal microscopy. Cytomembrane integrity and organelles damage were confirmed by detection of the reactive oxygen (ROS), lactate dehydrogenase (LDH), and mitochondrial membrane potential (Δψm). The underlying mechanism was manifested by Western blotting. The oncotic cell death was further confirmed by detection of oncosis related protein calpain. RESULTS: Swelling cell type and destroyed cytoskeleton were observed in QC2-treated HCC cells. Organelle damage was visualized by transmission electron microscopy. The detection of ROS accumulation, increased LDH release, and decreased ATP and Δψm confirmed the cell death. The oncotic related protein calpain was found to increase time-dependently in QC2-treated HCC cells, while its inhibitor PD150606 attenuated the cytotoxicity. CONCLUSIONS: Dehydroabietic acid derivative QC2 activated oncosis related protein calpain to induce the damage of cytomembrane and organelles which finally lead to oncosis in HCC cells.

Mechanisms of cataractogenesis in the presence of magnesium deficiency.

Senile cataract is the most common cause of bilateral blindness and results from the loss of transparency of the lens. Maintenance of the unique tissue architecture of the lens is vital for keeping the lens transparent. Membrane transport mechanisms utilizing several magnesium (Mg)-dependent ATPases, play an important role in maintaining lens homeostasis. Therefore, in Mg-deficiency states, ATPase dysfunctions lead to intracellular depletion of K(+) and accumulation of Na(+) and Ca(2+). High intracellular Ca(2+) causes activation of the enzyme calpain II, which leads to the denaturation of crystallin, the soluble lens protein required for maintaining the transparency of the lens. Mg deficiency also interferes with ATPase functions by causing cellular ATP depletion. Furthermore, Mg deficiency enhances lenticular oxidative stress by increased production of free radicals and depletion of antioxidant defenses. Therefore, Mg supplementation may be of therapeutic value in preventing the onset and progression of cataracts in conditions associated with Mg deficiency.

Expression of zinc-deficient human superoxide dismutase in Drosophila neurons produces a locomotor defect linked to mitochondrial dysfunction.

More than 130 different mutations in the Cu/Zn superoxide dismutase (SOD1) gene have been associated with amyotrophic lateral sclerosis but the mechanism of this toxicity remains controversial. To gain insight into the importance of the zinc site in the pathogenesis of SOD1 in vivo, we generated a Drosophila model with transgenic expression of a zinc-deficient human SOD1. Expression of zinc-deficient SOD1 in Drosophila resulted in a progressive movement defect with associated mitochondrial cristae vacuolization and reductions in adenosine triphosphate (ATP) levels. Furthermore, these flies are sensitized to mitochondrial toxins, paraquat, and zinc. Importantly, we show that the zinc-deficient SOD1-induced motor defect can be ameliorated by supplementing the endogenous fly respiratory chain machinery with the single-subunit NADH-ubiquinone oxidoreductase from yeast (NADH is nicotinamide adenine dinucleotide, reduced form.). These results demonstrate that zinc-deficient SOD1 is neurotoxic in vivo and suggest that mitochondrial dysfunction plays a critical role in this toxicity. The robust behavioral, pathological, and biochemical phenotypes conferred by zinc-deficient SOD1 in Drosophila have general implications for the role of the zinc ion in familial and sporadic amyotrophic lateral sclerosis.

Partial depletion of intracellular ATP mediates the stress-survival function of the PCPH oncoprotein.

Promotion of cellular resistance to stressful stimuli, including ionizing radiation and chemotherapeutic drugs, contributes to the transforming activity of the PCPH oncogene. The mechanism of this action, however, has remained unknown. Consistent with its intrinsic ATP diphosphohydrolase activity, expression of the PCPH oncoprotein in cultured cells has now been shown to result in partial depletion of intracellular ATP and consequent inhibition of the c-JUN NH2-terminal kinase-mediated stress signaling pathway. Supplementation of cells expressing the PCPH oncoprotein with exogenous ATP restored both stress-response signaling and sensitivity to cisplatin-induced apoptosis. In contrast, overexpression of the wild-type PCPH protein had a minimal effect on stress-induced signaling and on the cellular ATP content and did not protect cells from apoptosis. These results suggest that the PCPH oncoprotein confers resistance to stressors by reducing the cellular ATP concentration to levels below those required for optimal stress-induced signaling and apoptosis. Treatment with adenosine or nucleoside analogues may thus enhance the response to radiation or chemotherapy of tumors that express the PCPH oncogene.

Haemolytic anaemia in a patient with anorexia nervosa.

We report on a 17-year-old female patient with severe anorexia nervosa (AN) (body mass index of 9.8 kg/m(2)) who developed hypophosphataemia (serum phosphate 0.4 nmol/l) and subsequent haemolytic anaemia during oral refeeding. Hypophosphataemia due to an increased phosphate uptake may lead to a reduction of erythrocyte adenosine triphosphate. This mechanism is important for the differential diagnosis of haemolytic anaemia in patients with AN. To prevent this complication, phosphate supplementation should be considered in the refeeding of severely malnourished patients.

Anoxic ATP depletion in neonatal mice brainstem is prevented by creatine supplementation.

BACKGROUND: Sufficient ATP concentrations maintain physiological processes and protect tissue from hypoxic damage. With decreasing oxygen concentration, ATP synthesis relies increasingly on the presence of phosphocreatine. AIM: The effect of exogenously applied creatine on phosphocreatine and ATP concentrations was studied under control and anoxic conditions. METHODS: Pregnant mice were fed orally with creatine monohydrate (2 g/kg body weight/day). Brainstem slices from these mice pups were compared with those from pups of non-creatine supplemented pregnant mice. Measurements were performed under normoxic and anoxic conditions. In addition, brainstem slices from non-creatine treated mice pups were incubated for 3 hours in control artificial cerebrospinal fluid (CSF) (n = 10) or in artificial CSF containing 200 microM creatine (n = 10). ATP and phosphocreatine contents were determined enzymatically in single brainstem slices. RESULTS: ATP concentrations were in the same range in all preparations. However, there was a significant increase of phosphocreatine in the brainstems from pups of creatine fed mice when compared with the brainstems of pups from non-creatine treated mice or in non-incubated brainstems of control animals. After 30 minutes anoxia, ATP as well as phosphocreatine concentrations remained significantly higher in creatine pretreated slices compared with controls. CONCLUSION: The data indicate that exogenous application of creatine is effective in neuroprotection.

Improvement of cerebral ATP and choline deficiencies by Shao-Yin-Ren Shi-Quang-Da-Bu-Tang in senescence-accelerated mouse prone 8.

Shao-Yin-Ren Shi-Quang-Da-Bu-Tang (SDT) has been used traditionally to improve the systemic blood circulation and biological energy production in the body. The object of this study is to determine the effect of SDT extract on the decline of cerebral adenosine triphosphate (ATP) and choline content associated with learning and memory impairments in senescence-accelerated mice prone 8 (SAM P8). Twenty-four-week old mice were orally treated with SDT at 400 mg/kg body weight per day, and continued for 12 consecutive weeks. At the termination of the treatment, the body weight of SAM P8 was markedly lower than that of the equal aged senescence-resistant prone 1 (SAM R1), but this was conspicuously recovered to the level of SAM R1 by SDT treatment. SDT also significantly reduced the decline of cerebral weight (P < 0.05). By comparison with normal mice, a spontaneous decrease of cerebral ATP was observed in the SAM P8. Two- and 6-fold increases of cerebral ATP content were found in SAM R1 and SAM P8 by SDT administration, respectively. The cerebral choline content was significantly different between SAM R1 and SAM P8 aged 36-week old (P < 0.01). SDT remarkably restored the decrease of cerebral choline content in SAM P8 (P < 0.01). Taken together, these results demonstrate that SDT can reduce the decrease of cerebral weight, and restore the decline of cerebral ATP and choline content associated with an alteration of neuronal metabolism in SAM P8 brain. This suggests that pharmacological properties of SDT may participate in improvement of declined cerebral energy production and cholinergic neurotransmitter synthesis in senile dementia.

Graded ATP depletion can cause necrosis or apoptosis of cultured mouse proximal tubular cells.

The mechanisms of cell death induced by ATP depletion were studied in primary cultures of mouse proximal tubular (MPT) cells. Graded ATP depletion, ranging in severity from approximately 2 to 70% of control levels, was induced by incubating cells with either antimycin or 2-deoxyglucose, with varying concentrations of dextrose. We found that cells subjected to ATP depletion below approximately 15% of control died uniformly of necrosis. In contrast, cells subjected to ATP depletion between approximately 25 and 70% of control all died by apoptosis. The rapidity of cell death was proportional to the severity of reduction of cell ATP content and was independent of the mechanism of cell death. Renal growth factors, epidermal growth factor (EGF) and high-dose insulin, did not ameliorate apoptotic cell death induced by ATP depletion. We conclude that ATP depletion can cause either necrosis or apoptosis in MPT cells. Furthermore, we have identified a narrow range of ATP depletion (approximately 15 to 25% of control) representing a threshold that determines whether cells die by necrosis or apoptosis.

Decreased ATP cost of isometric contractions in ATP-depleted rat fast-twitch muscle.

ATP was depleted to inosine 5'-monophosphate (IMP) in gastrocnemius muscles of anesthetized rats during two consecutive bouts of intermittent tetanic stimulation (each 1 Hz at 100 Hz, 100-ms duration, for 3 min) separated by 5 min. During a subsequent 75-min recovery period, resynthesis of adenine nucleotide from IMP was inhibited in one group of muscles with hadacidin (N-formyl-N-hydroxyaminoacetate, total dose 300 mg/kg ip), an inhibitor of adenylosuccinate synthase. In vivo 31P-nuclear magnetic resonance spectra and analysis of muscle extracts confirmed that ATP was reduced by over 30% in hadacidin-treated muscles compared both with nonstimulated muscles and with identically stimulated and rested control muscles in saline-injected rats. After the recovery period, calculated [ADP] in ATP-depleted muscles was half that in controls, while phosphorylation potential [PP = ln([ATP]/[ADP] [Pi]), where Pi is inorganic phosphate] and twitch force were similar. During a final 8 min of 0.75-Hz twitch stimulation, [ADP] and the initial rate of phosphocreatine (PCr) hydrolysis were less in ATP-depleted compared with control muscles, while changes in PP and twitch force were similar in the two groups. During a final 3 min of tetanic stimulation, ATP, PCr, and pH decreased less in ATP-depleted compared with control muscles. The results indicate that the ATP cost of isometric contraction is decreased by acute ATP depletion.

Phosphorus deficiency and hypophosphatemia.

Low serum phosphorus levels, sometimes associated with depletion of phosphorus stores, can engender a variety of serious, often life-threatening physiologic changes. The proximate cause of this dangerous situation is usually medical intervention in such conditions as alcoholism and diabetic ketoacidosis, which can produce a shift of phosphorus within the body unless preventive measures are instituted.