Creatine and the control of energy metabolism in cardiac and skeletal muscle cells in culture

The addition of creatine to the culture media in which either cardiac or skeletal muscle cells were grown resulted in an increased concentration of intracellular phosphorylcreatine. Since the only metabolic path known for the synthesis of phosphorylcreatine is via creatine phosphokinase, it is suggested that the mitochondrial enzyme activity was stimulated by creatine, and that the ADP formed as a product of the reaction became available for the oxidative phosphorylation. The rate of synthesis of ATP must have been stimulated prior to the net increase in phosphorylcreatine. This finding provides evidence that creatine might be involved in a regulatory feedback mechanism maintaining the energy source for contraction and not serving merely as an energy store in the muscle.     

Serine signaling governs metabolic homeostasis and health

Serine has functions that are involved in metabolic homeostasis and health in pathological or stressful situations. Notably, the de novo serine synthesis pathway (SSP) plays a vital role in targeted regulation of immune responses, cell proliferation, and lipid/protein metabolism. The presentation of serine residues derived from SSP may be a signal of stress and provide novel insights into the relationship between metabolic homeostasis and diseases. Here, we summarize the current trends in understanding the regulatory mechanisms of serine metabolism, discuss how serine signaling governs metabolic and antistress processes, including oxidative stress, immunity, energy and lipid metabolism, intestinal microbiota, and the neurological system. We present a possible framework by which serine metabolism maintains metabolic homeostasis and treats human diseases.     

Amino acid metabolism by new-born rat heart cells in monolayer cultures

Heart cells from new-born rats grown in monolayer cultures took up extensively the essential amino acids provided by the culture medium for synthesizing proteins. Excessive utilization of glutamine and glutamic acid (Glx) and of arginine suggests that these amino acids are involved in intermediary metabolism. Moderate amounts of glycine and proline and high amounts of alanine are released into the culture medium. This release did not result from protein breakdown but represented de novo synthesis. Alanine synthesis was stimulated by extracellular glucose, pyruvate, Glx and serum, was not caused by hypoxia and did not correlate with glycolytic rates. It is suggested that de novo synthesis of alanine by myocardial cells is related to intracellular pyruvate concentration and has for primary function to feed the citric acid cycle with α ketoglutarate.     

Is greater tissue activity of creatine kinase the genetic factor increasing hypertension risk in black people of sub-Saharan African descent?

We postulate that the genetic factor increasing the propensity of black people of sub-Saharan African descent to develop high blood pressure is the relatively high activity of creatine kinase, predominantly in vascular and cardiac muscle tissue. Such greater activity of creatine kinase has been reported in skeletal muscle of black untrained subjects has been reported to be almost twice the activity found in white subjects. Creatine kinase, a key enzyme of cellular energy metabolism, increases the capacity of the cell to function under high demands. The enzyme regulates, buffers and transports, via phosphocreatine and creatine, energy produced by glycolysis and oxidative phosphorylation to sites of energy consumption such as myofibrils and membrane ion pumps. At these cellular locations, it is involved in the contraction process and active trans- membranous transport by readily providing the ATP needed for these processes. In addition, creatine kinase is increasingly reported to be involved in trophic responses. Furthermore, by using H+ and ADP to synthesize ATP, creatine kinase prevents acidification of the cell, providing relative protection against the effects of ischaemia. Greater creatine kinase activity in cardiovascular muscle and other tissues with high energy demands could increase cardiovascular contractile reserve, enhance trophic responses and increase renal tubular ability to retain salt. This could facilitate the development of arterial hypertension under chronic provocative circumstances, with higher mean blood pressures, more left ventricular hypertrophy and relatively fewer ischaemic events. Therefore, greater cellular activity of creatine kinase might explain the greater hypertension risk and the clinical characteristics of hypertensive disease observed in the black population.     

Individual variation in contractile cost and recovery in a human skeletal muscle.

This study determined the variation among individuals in ATP use during contraction and ATP synthesis after stimulation in a human limb muscle. Muscle energetics were evaluated using a metabolic stress test that separates ATP utilization from synthesis by 31P NMR spectroscopy. Epicutaneous supramaximal twitch stimulation (1 Hz) of the median and ulnar nerves was applied in combination with ischemia of the finger and wrist flexors in eight normal subjects. The linear creatine phosphate (PCr) breakdown during ischemic stimulation defined ATP use (delta PCr per twitch or approximately P/twitch) and was highly reproducible as shown by the relative standard deviation [(standard deviation/mean) x 100] of 11% in three repeated measures. The time constant of the monoexponential PCr change during aerobic recovery represented ATP synthesis rate and also showed a low relative standard deviation (9%). Individuals were found to differ significantly in both mean approximately P/twitch (PCr breakdown rates, 0.29-0.45% PCr per sec or % PCr per twitch; ANOVA, p < 0.001) and in mean recovery time constants (41-74 sec; ANOVA, P < 0.001). This range of approximately P/twitch corresponded with the range of fiber types reported for a flexor muscle. In addition, approximately P/twitch was negatively correlated with a metabolite marker of slow-twitch fiber composition (Pi/ATP). The nearly 2-fold range of recovery time constants agreed with the range of mitochondrial volume densities found in human muscle biopsies. These results indicate that both components involved in the muscle energy balance--oxidative capacity and contractile costs--vary among individuals in human muscle and can be measured noninvasively by 31 P NMR.     

De novo lipogenesis in health and disease

Background

De novo lipogenesis (DNL) is a complex and highly regulated metabolic pathway. In normal conditions DNL converts excess carbohydrate into fatty acids that are then esterified to storage triacylglycerols (TGs). These TGs could later provide energy via β-oxidation. In human body this pathway is primarily active in liver and adipose tissue. However, it is considered to be a minor contributor to the serum lipid homeostasis. Deregulations in the lipogenic pathway are associated with diverse pathological conditions.

Scope of review

The present review focuses on our current understanding of the lipogenic pathway with special reference to the causes and consequences of aberrant DNL.

Major conclusions

The deregulation of DNL in the major lipogenic tissues of the human body is often observed in various metabolic anomalies — including obesity, non-alcoholic fatty liver disease and metabolic syndrome. In addition to that de novo lipogenesis is reported to be exacerbated in cancer tissues, virus infected cells etc. These observations suggest that inhibitors of the DNL pathway might serve as therapeutically significant compounds. The effectiveness of these inhibitors in treatment of cancer and obesity has been suggested by previous works.

General significance

De novo lipogenesis – which is an intricate and highly regulated pathway – can lead to adverse metabolic consequences when deregulated. Therapeutic targeting of this pathway may open a new window of opportunity for combating various lipogenesis-driven pathological conditions — including obesity, cancer and certain viral infections.     

Influence of cellular energy metabolism on contractions of porcine carotid artery smooth muscle

A number of cellular metabolites, including inorganic phosphate and ADP, have been proposed to regulate the contractions of smooth muscle. Hypothesizing that one of these would have a greater influence than the others, parallel experiments using tissue mechanics and (31)P-NMR allowed comparison of several metabolic components with the generation of force in porcine carotid artery smooth muscle during long-term contractions. P(i), ADP, ATP, PCr, free energy, pH, and free Mg(2+) were determined from phosphate spectra during a control-hypoxia-postcontrol sequence generated during K(+) stimulation by replacement of oxygen with nitrogen using either pyruvate or glucose as substrate. Both pH and free Mg(2+) were significantly lower in control pyruvate-supplied tissues than in glucose-supplied tissues. Mechanical experiments following the same protocol produced variations in force. The pyruvate series produced the greater range of mechanical and metabolic changes. Linear and logarithmic regression analysis found the order of correlation with force to be highest for P(i), followed by pH, free energy, PCr, ATP, ADP, and free Mg(2+). The results are consistent with models for the regulation of myosin ATPase by free phosphate inhibition. The results are inconsistent with models of ADP as a regulator of smooth muscle force. Perturbations which alter intracellular phosphate, such as creatine loading, may produce side effects on the contractions of vascular smooth muscle.     

MR spectroscopy in heart failure—clinical and experimental findings

Magnetic resonance spectroscopy (MRS) allows for the non-invasive detection of a wide variety of metabolites in the heart. To study the metabolic changes that occur in heart failure, ³¹P- and ¹H-MRS have been applied in both patients and experimental animal studies. ³¹P-MRS allows for the detection of phosphocreatine (PCr), ATP, inorganic phosphate (Pi) and intracellular pH, while ¹H-MRS allows for the detection of total creatine. All these compounds are involved in the regulation of the available energy from ATP hydrolysis via the creatine kinase (CK) reaction. Using cardiac MRS, it has been found that the PCr/CK system is impaired in the failing heart. In both, patients and experimental models, PCr levels as well as total creatine levels are reduced, and in severe heart failure ATP is also reduced. PCr/ATP ratios correlate with the clinical severity of heart failure and, importantly, are a prognostic indicator of mortality in patients. In addition, the chemical flux through the CK reaction, measured with ³¹P saturation transfer MRS, is reduced more than the steady-state levels of high-energy phosphates in failing myocardium in both experimental models and in patients. Experimental studies suggest that these changes can result in increased free ADP levels when the failing heart is stressed. Increased free ADP levels, in turn, result in a reduction in the available free energy of ATP hydrolysis, which may directly contribute to contractile dysfunction. Data from transgenic mouse models also suggest that an intact creatine/CK system is critical for situations of cardiac stress. This work was supported by the British Heart Foundation and the Medical Research Council, London, England.     

Creatine and the Control of Myosin Synthesis in Differentiating Skeletal Muscle

These experiments provide evidence that creatine, an end product of contraction unique to muscle, is involved in the control of muscle-protein synthesis. Skeletal muscle cells formed both in vitro and in vivo synthesize myosin heavy chain faster when supplied creatine in vitro. The response is apparent within four hours after addition of creatine to the culture medium, and is dependent on concentration over a range of 10-100 muM creatine. The effect seems to be selective for cell-specific proteins(s), since the rate of total protein synthesis is unaffected.     

Regulation of ischemic muscle metabolism in peripheral arterial occlusive disease

A review of metabolic pathways is presented, which are involved in muscular energy production during hypoxia according to recent experimental findings. By means of own exercise examinations the course of reactions providing ATP anaerobically in the muscles of limbs with poor circulation is analysed. Therefore, the arteriovenous differences in the concentrations of lactate, pyruvate, ammonia, hypoxanthine and alanine in the femoral blood of patients with stage II AOD were determined. In addition, the intracellular phosphorus compounds ATP, PCr and Pi as well as the tissue pH were measured noninvasively in the calf muscles using 31P magnetic resonance spectroscopy. The results give evidence for marked activation of the creatine kinase reaction, of glycolysis, of the myokinase reaction and of the purine nucleotide cycle in the ischaemic musculature at loads of short duration, which are in total sufficient to maintain the concentration of ATP even during claudication pain. In spite of salvage pathways like alanine formation, the end products of these "emergency reactions", Pi, H+ and NH4+, accumulate and exert deleterious cytotoxic effects, which are thought to be responsible for rapid muscle fatigue and claudication pain in PAOD.     

Energy Metabolism is Compromised in Skeletal Muscle of Rats Chronically-Treated with Glutaric Acid

Glutaric acidemia type I (GA I) (GA I, McKusick 23167; OMIM # 231670) is an autosomal recessive metabolic disorder caused by glutaryl-CoA dehydrogenase deficiency (EC 1.3.99.7). Clinically, the disease is characterized by macrocephaly, hypotonia, dystonia and diskinesia. Since the pathophysiology of this disorder is not yet well established, in the present investigation we determined a number of energy metabolism parameters, namely ¹⁴CO₂ production, the activities of the respiratory chain complexes I–IV and of creatine kinase, in tissues of rats chronically exposed to glutaric acid (GA). High tissue GA concentrations (0.6 mM in the brain, 4 mM in skeletal muscle and 6 mM in plasma) were induced by three daily subcutaneous injections of saline-buffered GA (5 μmol·g⁻¹ body weight) to Wistar rats from the 5th to the 21st day of life. The parameters were assessed 12 h after the last GA injection in cerebral cortex and middle brain, as well as in skeletal muscle homogenates of GA-treated rats. GA administration significantly inhibited the activities of the respiratory chain complexes I-III and II and induced a significant increase of complex IV activity in skeletal muscle of rats. Furthermore, creatine kinase activity was also inhibited by GA treatment in skeletal muscle. In contrast, these measurements were not altered by GA administration in the brain structures studied. Taken together, it was demonstrated that chronic GA administration induced an impairment of energy metabolism in rat skeletal muscle probably due to a higher tissue concentration of this organic acid that may be possibly associated to the muscle weakness occurring in glutaric acidemic patients.     

Vitamin B6 is essential for serine <Emphasis Type="Italic">de novo</Emphasis> biosynthesis

Pyridoxal 5′-phosphate (PLP), the metabolically active form of vitamin B6, plays an essential role in brain metabolism as a cofactor in numerous enzyme reactions. PLP deficiency in brain, either genetic or acquired, results in severe drug-resistant seizures that respond to vitamin B6 supplementation. The pathogenesis of vitamin B6 deficiency is largely unknown. To shed more light on the metabolic consequences of vitamin B6 deficiency in brain, we performed untargeted metabolomics in vitamin B6-deprived Neuro-2a cells. Significant alterations were observed in a range of metabolites. The most surprising observation was a decrease of serine and glycine, two amino acids that are known to be elevated in the plasma of vitamin B6 deficient patients. To investigate the cause of the low concentrations of serine and glycine, a metabolic flux analysis on serine biosynthesis was performed. The metabolic flux results showed that the de novo synthesis of serine was significantly reduced in vitamin B6-deprived cells. In addition, formation of glycine and 5-methyltetrahydrofolate was decreased. Thus, vitamin B6 is essential for serine de novo biosynthesis in neuronal cells, and serine de novo synthesis is critical to maintain intracellular serine and glycine. These findings suggest that serine and glycine concentrations in brain may be deficient in patients with vitamin B6 responsive epilepsy. The low intracellular 5-mTHF concentrations observed in vitro may explain the favourable but so far unexplained response of some patients with pyridoxine-dependent epilepsy to folinic acid supplementation.     

Molecular mechanisms involved in hepatic steatosis and insulin resistance

Increased hepatic lipid content is associated with hepatic as well as whole‐body insulin resistance and is typical for individuals with type 2 diabetes mellitus. However, whether insulin resistance causes hepatic steatosis or whether hepatic steatosis per se reduces insulin sensitivity remains unclear. Multiple metabolic pathways lead to the development of hepatic steatosis, including enhanced free fatty acid release from adipose tissues (lipolysis), increased de novo fatty acid synthesis (lipogenesis), decreased mitochondrial β‐oxidation and decreased very low‐density lipoprotein secretion. Although the molecular mechanisms leading to the development of hepatic steatosis in the pathogenesis of type 2 diabetes mellitus are complex, several recent animal models have shown that modulating important enzymes involved in hepatic fatty acid and glycerolipid synthesis might be a key for treating hepatic insulin resistance. We highlight recent advances in the understanding of the molecular mechanisms leading to the development of hepatic steatosis and insulin resistance. (J Diabetes Invest, doi: 10.1111/j.2040‐1124.2011.00111.x, 2011)     

Frequent switching of Polycomb repressive marks and DNA hypermethylation in the PC3 prostate cancer cell line

Epigenetic reprogramming is commonly observed in cancer, and is hypothesized to involve multiple mechanisms, including DNA methylation and Polycomb repressive complexes (PRCs). Here we devise a new experimental and analytical strategy using customized high-density tiling arrays to investigate coordinated patterns of gene expression, DNA methylation, and Polycomb marks which differentiate prostate cancer cells from their normal counterparts. Three major changes in the epigenomic landscape distinguish the two cell types. Developmentally significant genes containing CpG islands which are silenced by PRCs in the normal cells acquire DNA methylation silencing and lose their PRC marks (epigenetic switching). Because these genes are normally silent this switch does not cause de novo repression but might significantly reduce epigenetic plasticity. Two other groups of genes are silenced by either de novo DNA methylation without PRC occupancy (5mC reprogramming) or by de novo PRC occupancy without DNA methylation (PRC reprogramming). Our data suggest that the two silencing mechanisms act in parallel to reprogram the cancer epigenome and that DNA hypermethylation may replace Polycomb-based repression near key regulatory genes, possibly reducing their regulatory plasticity.     

Modulation of hepatic steatosis by dietary fatty acids

Non-alcoholic fatty liver disease(NAFLD)describes a range of conditions caused by fat deposition within liver cells.Liver fat content reflects the equilibrium between several metabolic pathways involved in triglyceride synthesis and disposal,such as lipolysis in adipose tissue and de novo lipogenesis,triglyceride esterification,fatty acid oxidation and very-low-density lipoprotein synthesis/secretion in hepatic tissue.In particular,it has been demonstrated that hepatic de novo lipogenesis plays a significant role in NAFLD pathogenesis.It is widely known that the fatty acid composition of the diet influences hepatic lipogenesis along with other metabolic pathways.Therefore,dietary fat may not only be involved in the pathogenesis of hepatic steatosis,but may also prevent and/or reverse hepatic fat accumulation.In this review,major data from the literature about the role of some dietary fats as a potential cause of hepatic fat accumulation or as a potential treatment for NAFLD are described.Moreover,biochemical mechanisms responsible for an increase or decrease in hepatic lipid content are critically analyzed.It is noteworthy that both quantitative and qualitative aspects of dietary fat influence triglyceride deposition in the liver.A high-fat diet or the dietary administration of conjugated linoleic acids induced hepatic steatosis.In contrast,supplementation of the diet with krill oil or pine nut oil helped in the prevention and/or in the treatment of steatotic liver.Quite interesting is the"case"of olive oil,since several studies have often provided different and or conflicting results in animal models.     

Evaluation of myocardial energy status in vivo by NMR spectroscopy

Summary NMR spectroscopy is a powerful and non-invasive technique with which to study cardiac energy metabolism in vivo. This mcthod makes use of the "spin" properties of certain atomic nuclei. The naturally occurring phosphorus nucleus (P-31) is visible by NMR and phosphorus-31 NMR spectra contain signals from the major components of energy metabolism. In vivo, the phosphocreatine to ATP ratio (PCr/ATP) is used as an index of the energy status and viability of the myocardium. However, it is the response of this metabolic index to differing physiological and pharmacological stresses that has helped to elucidate the mechanisms that regulate cellular respiration and to highlight abnormalities in heart failure. As there are many technical difficulties involved with cardiac NMR, 31-phosphorus studies of skeletal muscle have provided an indirect way of studying abnormalities in myocardial metabolism in vivo.One of the unique features of NMR is that it permits in vivo measurements of fluxes through key enzymes in energy metabolism using magnetization transfer. Determination of the rates of energy transfer through the creatine kinase reaction and energy turnover in vivo will provide new insights into the control of energy metabolism in health and disease. Alternatively, carbon-13 NMR can be used to measure fluxes through the different metabolic pathways of synthesis and catabolism following administration of selectively labelled carbon-13 substrates. In conclusion, the non-invasive and versatile nature of NMR spectroscopy makes it an ideal method to assess and evaluate energy metabolism in vivo.     

Skeletal muscle ATP kinetics are impaired in frail mice

The interleukin-10 knockout mouse (IL10^tm/tm) has been proposed as a model for human frailty, a geriatric syndrome characterized by skeletal muscle (SM) weakness, because it develops an age-related decline in SM strength compared to control (C57BL/6J) mice. Compromised energy metabolism and energy deprivation appear to play a central role in muscle weakness in metabolic myopathies and muscular dystrophies. Nonetheless, it is not known whether SM energy metabolism is altered in frailty. A combination of in vivo ³¹P nuclear magnetic resonance experiments and biochemical assays was used to measure high-energy phosphate concentrations, the rate of ATP synthesis via creatine kinase (CK), the primary energy reserve reaction in SM, as well as the unidirectional rates of ATP synthesis from inorganic phosphate (P_i) in hind limb SM of 92-week-old control (n = 7) and IL10^tm/tm (n = 6) mice. SM Phosphocreatine (20.2 ± 2.3 vs. 16.8 ± 2.3 μmol/g, control vs. IL10^tm/tm, p < 0.05), ATP flux via CK (5.0 ± 0.9 vs. 3.1 ± 1.1 μmol/g/s, p < 0.01), ATP synthesis from inorganic phosphate (P_i → ATP) (0.58 ± 0.3 vs. 0.26 ± 0.2 μmol/g/s, p < 0.05) and the free energy released from ATP hydrolysis (∆G_∼ATP) were significantly lower and [P_i] (2.8 ± 1.0 vs. 5.3 ± 2.0 μmol/g, control vs. IL10^tm/tm, p < 0.05) markedly higher in IL10^tm/tm than in control mice. These observations demonstrate that, despite normal in vitro metabolic enzyme activities, in vivo SM ATP kinetics, high-energy phosphate levels and energy release from ATP hydrolysis are reduced and inorganic phosphate is elevated in a murine model of frailty. These observations do not prove, but are consistent with the premise, that energetic abnormalities may contribute metabolically to SM weakness in this geriatric syndrome.     

Induction of cyclooxygenase protein and stimulation of prostaglandin E2 release by epidermal growth factor in cultured guinea pig gastric mucous cells

The present study was undertaken to investigate whether epidermal growth factor (EGF) could stimulate prostaglandin E₂ release, and if so, by what mechanism EGF would exert such an effect in gastric mucosal cells. In cultured guinea pig gastric mucous cells, EGF dosedependently stimulated prostaglandin E₂ release, with maximal stimulation observed at 10 ng/ml. EGF stimulated an increase in cyclooxygenase activity, which was reduced by protein synthesis inhibitor, actinomycin D, and cycloheximide. EGF also stimulated the enzyme protein synthesis estimated by Western blot analysis, whereas EGF did not stimulate phospholipase A₂ activity. These results suggest that such an effect of EGF onde novo synthesis of cyclooxygenase protein and prostaglandin E₂ release may be involved at least in part in the mechanism of EGF-induced local regulation of gastric mucosal integrity.