Acid Substrate Cycling Research Articles

Increased triacylglycerol - Fatty acid substrate cycling in human skeletal muscle cells exposed to eicosapentaenoic acid.

It has previously been shown that pretreatment of differentiated human skeletal muscle cells (myotubes) with eicosapentaenoic acid (EPA) promoted increased uptake of fatty acids and increased triacylglycerol accumulation, compared to pretreatment with oleic acid (OA) and palmitic acid (PA). The aim of the present study was to examine whether EPA could affect substrate cycling in human skeletal muscle cells by altering lipolysis rate of intracellular TAG and re-esterification of fatty acids. Fatty acid metabolism was studied in human myotubes using a mixture of fatty acids, consisting of radiolabelled oleic acid as tracer (14C-OA) together with EPA or PA. Co-incubation of myotubes with EPA increased cell-accumulation and incomplete fatty acid oxidation of 14C-OA compared to co-incubation with PA. Lipid distribution showed higher incorporation of 14C-OA into all cellular lipids after co-incubation with EPA relative to PA, with most markedly increases (3 to 4-fold) for diacylglycerol and triacylglycerol. Further, the increases in cellular lipids after co-incubation with EPA were accompanied by higher lipolysis and fatty acid re-esterification rate. Correspondingly, basal respiration, proton leak and maximal respiration were significantly increased in cells exposed to EPA compared to PA. Microarray and Gene Ontology (GO) enrichment analysis showed that EPA, related to PA, significantly changed i.e. the GO terms “Neutral lipid metabolic process” and “Regulation of lipid storage”. Finally, an inhibitor of diacylglycerol acyltransferase 1 decreased the effect of EPA to promote fatty acid accumulation. In conclusion, incubation of human myotubes with EPA, compared to PA, increased processes of fatty acid turnover and oxidation suggesting that EPA may activate futile substrate cycling of fatty acids in human myotubes. Increased TAG—FA cycling may be involved in the potentially favourable effects of long-chain polyunsaturated n-3 fatty acids on skeletal muscle and whole-body energy metabolism.

Submolecular regulation of cell transformation by deuterium depleting water exchange reactions in the tricarboxylic acid substrate cycle

The naturally occurring isotope of hydrogen (1H), deuterium (2H), could have an important biological role. Deuterium depleted water delays tumor progression in mice, dogs, cats and humans. Hydratase enzymes of the tricarboxylic acid (TCA) cycle control cell growth and deplete deuterium from redox cofactors, fatty acids and DNA, which undergo hydride ion and hydrogen atom transfer reactions. A model is proposed that emphasizes the terminal complex of mitochondrial electron transport chain reducing molecular oxygen to deuterium depleted water (DDW); this affects gluconeogenesis as well as fatty acid oxidation. In the former, the DDW is thought to diminish the deuteration of sugar-phosphates in the DNA backbone, helping to preserve stability of hydrogen bond networks, possibly protecting against aneuploidy and resisting strand breaks, occurring upon exposure to radiation and certain anticancer chemotherapeutics. DDW is proposed here to link cancer prevention and treatment using natural ketogenic diets, low deuterium drinking water, as well as DDW production as the mitochondrial downstream mechanism of targeted anti-cancer drugs such as Avastin and Glivec. The role of 2H in biology is a potential missing link to the elusive cancer puzzle seemingly correlated with cancer epidemiology in western populations as a result of excessive 2H loading from processed carbohydrate intake in place of natural fat consumption.

Effect of cancer cachexia on triacylglycerol/fatty acid substrate cycling in white adipose tissue

The effect of cancer cachexia on the TAG/FA substrate cycle in white adipose tissue was determined in vivo using the MAC16 murine model of cachexia. When compared with non-tumor-bearing animals, the rate of TAG-glycerol production was found to be increased almost threefold in animals bearing the MAC13 tumor, which does not induce cachexia, but was not further elevated in animals bearing the MAC16 tumor. In both cases TAG-glycerol production and de novo synthesis of TAG-FA were also increased above non-tumor-bearing animals. In animals bearing the MAC16 tumor, the TAG-FA rates were significantly higher than in animals bearing the MAC13 tumor. This suggests that the presence of the tumor alone is sufficient to cause an increase in cycling rate, and in the absence of an elevated energy intake (MAC16) this may contribute to the depletion of adipose tissue.

The regulatory, informational, and immunomodulatory roles of fat fuels

Fat oxidation provides a fuel for many tissues and it provides an important signal to decrease glucose utilization and oxidation in muscle and so conserve glucose for essential organs such as the brain. The control of fatty acid oxidation is achieved in part through its plasma concentrations, which may be precisely controlled by the triacylglycerol-fatty acid substrate cycle, which can also, if oxidation is taken into account, be viewed as a branch point in this important pathway. Branch points may provide precision in regulation if one of the fluxes at the branch is low compared with the other flux. Both branch points and substrate cycles are energetically expensive and may account for some of the increases in energy expenditure in conditions of injury, burns, and sepsis and in the postexercise condition. Fatty acids, through effects on plasma free tryptophan concentrations and hence 5-hydroxytryptamine concentrations in the brain, may play a role in central fatigue. Polyunsaturated fatty acids are claimed to have immunosuppressive properties. Work has been done to provide a biochemical analysis of how they might influence some functions of cells of the immune system.

Sex Difference in Triglyceride/Fatty Acid Substrate Cycling of Rat Adipose Tissue: Indirect Regulation by Androgens

Male Sprague-Dawley rats displayed significantly higher rates of triglyceride/fatty acid (TG/FFA) substrate cycling in subcutaneous, perigenital, and mesenteric white adipose tissue, compared to females. To investigate possible regulation via androgens and estrogens, male rats were treated with the androgen antagonist, cyproterone acetate (10 mg daily in subcutaneous injections), or estradiol polyphosphate (0.3 mg intramuscularly, given as a single dose). Estradiol treatment did not affect TG/FFA cycling. Treatment with cyproterone acetate significantly decreased TG/FFA cycling in perigenital (epididymal) tissue. This effect could however largely be ascribed to concomitant inhibition of food intake by cyproterone acetate. The effects of cyproterone acetate on the two axes of TG/FFA cycling (lipolysis and re-esterification) were further studied in vitro. Norepinephrine-stimulated glycerol release from perigenital adipocytes was inhibited, whereas activities of esterification enzymes (GPAT and PPH) was essentially unaffected. We conclude that androgens seem to affect TG/FFA cycling indirectly via the lipolytic axis.

Lactate metabolism in inactive skeletal muscle during lactacidosis

Contributions of carbohydrate and fat metabolism to the removal of a lactate (Lac-) load were quantified in inactive soleus (SL), plantaris (PL), and white gastrocnemius (WG) rat hindlimb muscle. Male Sprague-Dawley rats were perfused for 60 min with normal perfusate (NP, n = 8) or a high-lactate perfusate (LP, n = 8), simulating ionic conditions found in arterial blood and plasma after intense exercise: Lac- = 11.0 mM, K+ = 7.88 mM, and pH = 7.15. Metabolite fluxes across the hindlimb were calculated from blood flow and arteriovenous differences. In NP, Lac- was continuously released (2.9 +/- 0.2 mumol.min-1 x 100 g-1). However, in LP, a rapid and significant uptake of Lac- increased muscle Lac- fivefold to 39.6 +/- 1.1, 33.1 +/- 2.2, and 28.8 +/- 1.7 mumol/g dry wt in SL, PL, and WG, respectively. Glucose and O2 uptakes were similar during LP and NP perfusion. Glycerol release increased eightfold to 3.3 +/- 0.7 mumol.min-1 x 100 g-1 in response to LP. Muscle ATP, creatine phosphate, glycogen, glycolytic intermediate, and triacylglycerol concentrations did not change. However, muscle lactate-to-pyruvate ratios were elevated in all muscles of the LP group postperfusion, indicating changes in the mass action ratio at the pyruvate dehydrogenase reaction. In LP, of 80 mumol of Lac- taken up, 11% was accounted for by increased muscle Lac-, 12-24% was oxidized, and 5% may have been involved in glycerol release. The remaining Lac- may have been involved in metabolic cycling along the glyconeogenic-glycolytic pathway and/or in triacylglycerol-free fatty acid substrate cycling.

Circulating metabolite utilization by periuterine adipose tissue in situ in the pregnant rat

To study the use of glucose for lipid synthesis by the periuterine adipose tissue in situ, 14C-glucose was infused through the left uterine artery of anesthetized, fed pregnant and virgin control rats. A greater amount of 14C-lipid always appeared in the adipose tissue from the left uterine horn than in the tissue from the right uterine horn, indicating direct utilization of the infused 14C-glucose by the tissue. Glucose utilization for both glyceride glycerol and fatty acid synthesis increased from day 0 (virgin rats) to day 20 of gestation and then decreased dramatically on day 21. In virgin and 12-day pregnant rats, glucose was incorporated into either lipidic moiety at similar rates, whereas in late pregnant rats glucose utilization for glyceride glycerol synthesis was four to five times greater than for fatty acids. The utilization of circulating fatty acids and the lipoprotein triglyceride-derived fatty acids was studied by infusing 14C-palmitate or 14C-triolein-labeled very-low-density lipoprotein (VLDL) through the left uterine artery in both virgin and 20-day pregant rats. Incorporation of fatty acids from either one of these plasma sources was significantly higher in the pregnant than in virgin rats. This high amount of fatty acid acquisition did not account for the very active glyceride glycerol synthesis observed in pregnant rats and can only be explained by the intracellular reesterification of some lipolitic fatty acids. The results suggest a highly accelerated triacylglycerol/fatty acid substrate cycle in adipose tissue during late pregnancy, which would allow active esterification (contributing to fat accumulation) and responsive lipolysis (permitting rapid fat mobilization) by the mother.

Tissue-specific effects of rapid tumour growth on lipid metabolism in the rat during lactation and on litter removal

1. The effect of tumour burden on lipid metabolism was examined in virgin, lactating and litter-removed rats. 2. No differences in food intake or plasma insulin concentrations were observed between control animals and those bearing the Walker-256 carcinoma (3-5% of body wt.) in any group studied. 3. In virgin tumour-bearing animals, there was a significant increase in liver mass, blood glucose and lactate, and plasma triacylglycerol; the rate of oxidation of oral [14C]lipid to 14CO2 was diminished, and parametrial white adipose tissue accumulated less [14C]lipid compared with pair-fed controls. 4. These findings were accompanied by increased accumulation of lipid in plasma and decreased white-adipose-tissue lipoprotein lipase activity. 5. In lactating animals, tumour burden had little effect on the accompanying hyperphagia or on pup weight gain; tissue lipogenesis was unaffected, as was tissue [14C]lipid accumulation, plasma [triacylglycerol] and white-adipose-tissue and mammary-gland lipoprotein lipase activity. 6. On removal (24 h) of the litter, the presence of the tumour resulted in decreased rates of lipogenesis in the carcass, liver and white and brown adipose tissue, decreased [14C]lipid accumulation in white adipose tissue, but increased accumulation in plasma and liver, increased plasma [triacylglycerol] and decreased lipoprotein lipase activity in white adipose tissue. 7. The rate of triacylglycerol/fatty acid substrate cycling was significantly decreased in white adipose tissue of virgin and litter-removed rats bearing the tumour, but not in lactating animals. 8. These results demonstrate no functional impairment of lactation, despite the presence of tumour, and the relative resistance of the lactating mammary gland to the disturbance of lipid metabolism that occurs in white adipose tissue of non-lactating rats with tumour burden.

The influence of starvation and natural refeeding on the rate of triacylglycerol/fatty acid substrate cycling in brown adipose tissue and different white adipose sites of the rat in vivo. The role of insulin and the sympathetic nervous system.

Triacylglycerol/fatty acid substrate cycling was measured in vivo in brown adipose tissue (BAT) and white adipose tissue (WAT) of fed, starved and refed rats. Starvation (24h) significantly decreased the rate of cycling in BAT, and refeeding chow diet led to a rapid, 6-fold increase in cycling. Cycling rate in WAT was much lower than in BAT, and was not influenced by fasting or refeeding. Similar rates of cycling were found in epididymal, mesenteric, subcutaneous, and scapular WAT depots. Sympathetic denervation of interscapular BAT abolished the response of the tissue to refeeding, as did acute suppression of insulin secretion. Similarly, rats fasted for 3 days showed no acute increase in the activity of the cycle following refeeding.

Effects of lactation and removal of pups on the rate of triacyglycerol/fatty acid substrate cycling in white adipose tissue of the rat

The rate of the triacylglycerol/fatty acid substrate cycle was measured in vivo in adipose tissue of virgin and lactating rats with pups removed. The rate decreased by 70% in adipose tissue of lactating rats and increased 9-fold on removal of the pups. Similar differences in cycling rate were seen in adipose tissue incubated in vitro in the presence of isoprenaline.

Lipolytic response to glucose infusion in human subjects.

We have determined the effect of various rates of glucose infusion on the rates of release of glycerol (Ra glycerol), free fatty acids (Ra FFA), and on energy metabolism in normal human volunteers. Plasma kinetics were determined with use of the stable isotopic tracers D-5-glycerol and [1-13C]palmitate, and energy metabolism was determined by indirect calorimetry. The effect of glucose infusion on Ra glycerol and Ra FFA was dose-dependent. At an infusion rate of 1 mg X kg-1 X min-1, no effect on either kinetic factor was noted. At 4 mg X kg-1 X min-1, both Ra glycerol and Ra FFA were suppressed; at 8 mg X kg-1 X min-1, Ra FFA was even more depressed, but Ra glycerol was similar to the value during the 4 mg X kg-1 X min-1 infusion. At all infusion rates tested, the amount of potential energy available from the sum of the glucose infusion and endogenously mobilized fat was always greater than when no glucose was infused. Glucose decreased fat mobilization by both inhibiting lipolysis and stimulating reesterification, thus causing a significant increase in triglyceride-fatty acid substrate cycling within the adipose tissue.

Effects of hormones on the rate of the triacylglycerol/fatty acid substrate cycle in adipocytes and epididymal fat pads

Brian Brooks, Jonathan R.S. Arch + and Eric A. Newsholme* Department of Biochemistry, University of Oxford, South Parks Road, Oxford OX1 3QU and + Beecham Pharmaceuticals Research Division, Biosciences Research Centre, Great Burgh, Yew Tree Bottom Road, Epsom, Surrey KT18 5XQ, England Received 12 July 1982 Hormone Triacylglycerol/ fatty acid substrate cycle Substrate cycling Noradrenaline Glucagon Triacylglycerolsynthesis 1. INTRODUCTION It has been suggested by some workers that sub- strate ('futile') cycles are either of little of no sig- nificance in metabolism [1] whereas others suggest that they are of considerable importance in in- creasing sensitivity in metabolic control [2-4]. One prediction of the hypothesis that cycling increases sensitivity is that the rate will vary from one con- dition to another and that some hormones may specifically increase the cycling rate. Evidence for the existence of the triacylglycerol/fatty acid cycle in adipose tissue was presented by Steinberg [5] who compared the rates of fatty acid and glycerol production by isolated epididymal fat pads. This method has been used to investigate the effect of some hormones on the rate of the triacylglycerol/ fatty acid cycle in isolated adipocytes of the rat. In addition, a radiochemical method that depends upon the incorporation of tritium from tritiated water into the glycerol and fatty acid moieties of triacylglycerol has been developed for the mea- surement of the rate of this cycle in epididymal fat pads of the rat. The effects of some hormones have been investigated using both techniques and the results are reported and discussed here. 2. MATERIALS AND METHODS Male Wistar rats were obtained from OLAC (1976) Ltd. (Blackthorn, Bicester, Oxon OX60TP). * To whom correspondence should be addressed Chemicals, biochemicals and enzymes were ob- tained from sources given in [6] except that crys- talline glucagon, ACTH and TSH were obtained from Sigma Chemical Co. (Poole, Dorset) and radiochemicals from The Radiochemical Centre (Amersham HP7 9LL). Rats were killed by cervical dislocation and the epididymal fat pads were removed. Only the thin- ner distal portion of the pad was used for incuba- tion and up to 200 mg fresh tissue was incubated in 1.5 ml Krebs-Ringer bicarbonate buffer con- taining 1 mM glucose and 4% (w/v) defatted al- bumin [6]. Fat cells were prepared as in [7] and in- cubated in 1.3 ml Krebs-Ringer buffer as in [6]. The incubation was terminated by addition of 300/tl 2 M H2SO4 (which does not precipitate al- bumin); 200 ttl was taken for assay of fatty acid and 1 ml for assay of glycerol. For the latter, the cells and albumin were precipitated by addition of trichloroacetic acid (200/d, 40% (w/v)). The pre- cipitate was removed by centrifugation and univer- sal indicator (15/~1) was added to the supernatant and neutralised by addition of 7 M KOH from a microsyringe. The concentrations of glycerol and fatty acids were determined as in [8,9]. The rate of cycling was calculated as follows: cycling -- [(3 x rate of glycerol production)