Exogenous Substrate Utilization Research Articles

Integration of parallel metabolomics and transcriptomics reveals metabolic patterns in porcine oocytes during maturation.

Well-controlled metabolism is the prerequisite for optimal oocyte development. To date, numerous studies have focused mainly on the utilization of exogenous substrates by oocytes, whereas the underlying mechanism of intrinsic regulation during meiotic maturation is less characterized. Herein, we performed an integrated analysis of parallel metabolomics and transcriptomics by isolating porcine oocytes at three time points, cooperatively depicting the global picture of the metabolic patterns during maturation. In particular, we identified the novel metabolic features during porcine oocyte meiosis, such as the fall in bile acids, the active one-carbon metabolism and a progressive decline in nucleotide metabolism. Collectively, the current study not only provides a comprehensive multiple omics data resource, but also may facilitate the discovery of molecular biomarkers that could be used to predict and improve oocyte quality.

Effects of Long-Term Microgravitation Exposure on Cell Respiration of the Rat Musculus Soleus Fibers

Cell respiration of the m. soleus fibers was studied in Wistar rats treated with succinic acid and exposed to microgravitation for 35 days. The results indicated that respiration rates during utilization of endogenous and exogenous substrates and the maximum respiration rate decreased in animals subjected to microgravitation without succinate treatment. The respiration rate during utilization of exogenous substrate did not increase in comparison with that on endogenous substrates. Succinic acid prevented the decrease in respiration rate on endogenous substrates and the maximum respiration rate. On the other hand, the respiration rate on exogenous substrates was reduced in vivarium control rats receiving succinate in comparison with intact control group. That could indicate changed efficiency of complex I of the respiratory chain due to reciprocal regulation of the tricarbonic acid cycle.

No Sex Differences in Substrate Utilization during Exercise in Healthy Adolescents

Compared with men, women utilize relatively more fat and less endogenous carbohydrate (CHOendo), but preferentially oxidize exogenous CHO (CHOexo) during exercise. As it has been demonstrated that pre- and early-pubertal children do not exhibit sex differences in substrate utilization, the adult phenotype is likely due to sex hormones. Whether sex influences substrate utilization during exercise in adolescents, in whom differences in sex hormones become larger, is unclear. PURPOSE: To determine the influence of sex on endogenous and exogenous substrate utilization in healthy adolescent boys and girls. METHODS: Twenty-three 14-yr-old boys (n=13) and regularly menstruating girls (n=10) participated. Boys were taller (p=0.01) and had significantly less body fat (p=0.006), but both sexes had equal aerobic fitness levels (ml O2/min) expressed relative to fat-free mass (FFM; mean ± SEM; 53.1 ± 1.5 and 51.8 ± 2.0 for boys and girls, respectively). All subjects cycled for 60 min at ∼70% VO2max while intermittently drinking a 13C-enriched 6 % CHOexo (4 % sucrose, 2 % glucose) solution (∼1.5g/kg) in one trial (CT) or flavoured water in another (WT). Both trials were completed by the girls within the follicular phase of their menstrual cycle. RER and 13C/12C ratio in expired CO2 were determined during the last 15 min of exercise. RESULTS: RER was lower (p=0.007) during WT (0.95 ± 0.01) than CT (0.97 ± 0.01), with no difference between sexes (p=0.9). CHOexo oxidation rates (in mg/ kgFFM/min) were similar (p=0.72) between boys and girls (9.1 ± 0.5 and 8.8 ± 0.5, respectively), as was the % contribution from CHOexo to total energy yield (18.6 ± 0.8 and 18.6 ± 1.1, respectively; p=0.9) and the oxidation ratio of CHOexo: CHOendo (0.25 ± 0.01 and 0.26 ± 0.02, respectively; p=0.61). CONCLUSIONS: These results demonstrate that during adolescence, sex is not a major determinant of fuel selection during exercise, at least of relatively high intensity. One implication might be that adolescent boys and girls can equally derive metabolic and performance benefits from consuming CHO during competition.

Glycerylprostaglandin Synthesis by Resident Peritoneal Macrophages in Response to a Zymosan Stimulus

Cyclooxygenase (COX)-2 oxygenates arachidonic acid (AA) and 2-arachidonylglycerol (2-AG) to endoperoxides, which are subsequently transformed to prostaglandins (PGs) and glycerylprostaglandins (PG-Gs). PG-G formation has not been demonstrated in intact cells treated with a physiological agonist. Resident peritoneal macrophages, which express COX-1, were pretreated with lipopolysaccharide to induce COX-2. Addition of zymosan caused release of 2-AG and production of the glyceryl esters of PGE2 and PGI2 over 60 min. The total quantity of PG-Gs (16 +/- 6 pmol/10(7) cells) was much lower than that of the corresponding PGs produced from AA (21,000 +/- 7,000 pmol/10(7) cells). The differences in PG-G and PG production were partially explained by differences in the amounts of 2-AG and AA released in response to zymosan. The selective COX-2 inhibitor, SC236, reduced PG-G and PG production by 49 and 17%, respectively, indicating a significant role for COX-1 in PG-G and especially PG synthesis. Time course studies indicated that COX-2-dependent oxygenation rapidly declined 20 min after zymosan addition. When exogenous 2-AG was added to macrophages, a substantial portion was hydrolyzed to AA and converted to PGs; 1 microm 2-AG yielded 820 +/- 200 pmol of PGs/10(7) cells and 78 +/- 41 pmol of PG-Gs/10(7) cells. SC236 reduced PG-G and PG production from exogenous 2-AG by 88 and 76%, respectively, indicating a more significant role for COX-2 in the utilization of exogenous substrate. In conclusion, lipopolysaccharide-pretreated macrophages produce PG-Gs from endogenous 2-AG during zymosan phagocytosis, but PG-G formation is limited by substrate hydrolysis and inactivation of COX-2.

Effects of different oxidative insults on intermediary metabolism in isolated perfused rat hearts

13C and 31P NMR were used to evaluate exogenous substrate utilization and endogenous phosphate metabolites in perfused rat hearts exposed to tert-butylhydroperoxide ( tert-BOOH) and hydrogen peroxide (H 2O 2). Both reagents caused a reduction in developed pressure compared to controls and, in agreement with previous 31P NMR data, 8 had different effects on intracellular high-energy phosphates and glycolysis. 13C Isotopomer analysis of tissue extracts showed that H 2O 2 and tert-BOOH also had significantly different effects on substrate utilization by the citric acid cycle. The contribution of exogenous lactate and glucose to acetyl-CoA was 43% in controls and increased to over 80% in the presence of either oxidant. With tert-BOOH, exogenous glucose and lactate were both significant contributors to acetyl-CoA (44 ± 2 and 41 ± 3%). However, with H 2O 2, exogenous lactate supplied a much higher fraction of acetyl-CoA (72 ± 2%) than glucose (9 ± 1%). Also, when [2- 13C]glucose was supplied, accumulation of [2- 13C] and [5- 13C]fructose 1,6-bisphosphate was observed in the presence of H 2O 2, indicating inhibition of glyceraldehyde-3-phosphate dehydrogenase. These results indicate that despite this glycolytic inhibition, H 2O 2 increased the utilization of pyruvate precursors when lactate was present as an alternative carbohydrate substrate.

Temporal analysis of myocardial glucose metabolism by 2-[18F]fluoro-2-deoxy-D-glucose

To assess kinetic changes of myocardial glucose metabolism after physiological interventions, we perfused isolated working rat hearts with glucose and 2-[18F]fluoro-2-deoxy-D-glucose (2-FDG). Tissue uptake of 2-FDG and the input function were measured on-line by external detection. The fractional rate of 2-FDG phosphorylation was determined by graphical analysis of time-activity curves. The steady-state uptake of 2-FDG was linear with time, and the tracer was retained predominantly in its phosphorylated form. Tissue accumulation of 2-FDG decreased with a reduction in work load and with the addition of competing substrates. Insulin caused a significant increase in 2-FDG accumulation in hearts from fasted but not from fed animals. We conclude that in the isolated working rat heart there is rapid adjustment of exogenous substrate utilization and that most interventions known to alter glucose metabolism induce parallel changes in 2-FDG uptake. Qualitative differences in the in vitro response to insulin may be affected by the presence of either endogenous insulin or glycogen.

A quantitative evaluation of stress associated with indwelling superior vena cava cannulae in rats

“Nonrestraining” superior vena cava (SVC) cannulae are commonly used in rat metabolic studies. The objective of this study was to evaluate the level of stress and metabolic alterations associated with surgical placement and subsequent maintenance and use of an indwelling intravenous cannula as measured by resting energy expenditure (REE), respiratory quotient (RQ), urinary nitrogen excretion, and substrate utilization pattern. Nine rats underwent SVC cannulation and six rats underwent sham operation. Postoperatively, animals were starved for 48 hr and then refed either parenterally or orally for 48 hr. Sham-operated animals adapted appropriately to starvation within 24 hr by decreasing REE 14% and increasing utilization of fat (RQ:0.89 → 0.78). Hypometabolic adaptation to starvation was delayed in cannulated animals until the second postoperative day, but appropriate alterations in energy substrate utilization were not affected by the presence of cannulae. Cannulae did not affect the metabolic response to oral refeeding with similar increases in REE and similar patterns of substrate utilization in sham-operated and cannulated animals. Animals refed parenterally demonstrated higher REE and apparent fat deposition (RQ > 1) consistent with continuous hypercaloric glucose administration. Urinary nitrogen excretion was not affected by the presence of cannulae. Cannula-associated metabolic alterations are minimal and transient and do not preclude detection and quantification of alterations associated with composition or utilization of exogenous substrates.

Arachidonic acid and prostaglandin endoperoxide metabolism in isolated rabbit and coronary microvessels and isolated and cultivated coronary microvessel endothelial cells.

Isolated microvessels and isolated and cultured microvessel endothelial cells were prepared from rabbit cardiac muscle. Pathways of arachidonic acid metabolism were determined by measurement of exogenous substrate utilization [( 1-14C]arachidonic acid incorporation and release from intact tissue and cells; [1-14C]prostaglandin H2 (PGH2) metabolism by broken cell preparations) and by quantification of endogenous products (immunoreactive 6-keto-prostaglandin F1 alpha (PGF1 alpha) and prostaglandin E (PGE) release) by selective radioimmunoassay. Rabbit coronary microvessels and derived microvascular endothelial cells (RCME cells) synthesized two major products of the cyclooxygenase pathway: 6-keto-PGF1 alpha (hydrolytic product of prostaglandin I2) and PGE2. A reduced glutathione requiring PGH-E isomerase was demonstrated in coronary microvessels and RCME cells, but not in rabbit circumflex coronary artery or aorta. In addition, a minor amount of a compound exhibiting similar characteristics to 6-keto-PGE1 was found to be produced by microvessels and RCME cells. Measurement of endogenously released prostaglandins indicated that under basal and stimulated conditions, PGE release exceeded that of 6-keto-PGF1 alpha. Microvessels and microvessel endothelial cells derived from cardiac muscle of rabbit exhibit pathways of arachidonate metabolism that are different from those of many large blood vessels and derived endothelial cells.

Kinetin-Mediated Stimulation of Accumulation of Buckwheat Flavonoids in the Dark

A short treatment of excised buckwheat cotyledons with a solution of kinetin lead to an up to 9-fold stimulation of anthocyanin biosynthesis, to an about 50 percent increase in the accumula­tion of rutin, and to an about 30 percent increase, on the average, in the accumulation of C-glycosylflavones in the treated material during its posttreatment incubation in the dark. When the treated cotyledons were incubated in a solution of ʟ--phenylalanine anthocyanin accumulation in the dark practically attained the same high level as it was observed in the illuminated cotyledons fed with exogenous ʟ--phenylalanine. In experiments with l4C-labelled L-phenylalanine kinetin induced a sharp rise in the labelling (resp. in the utilization of exogenous substrate for biosynthesis) of anthocyanins and rutin in the dark and a slight increase in the radioactivity of C-glycosylflavones. Similar labelling changes occurred in the illuminated cotyledons. However, both kinetin and light still more effectively promoted biosynthetic use of the endogenous sub­strate. As a result the relative portion of flavonoids formed from exogenous L-phenylalanine under these conditions showed a decrease as compared with the ratio of precursor use in the un­treated cotyledons. The results show that low accumulation rates of anthocyanins and other flavo­noids in the dark are conditioned by the limited access of substrate (ʟ--phenylalanine) molecules to the flavonoid enzymes lending further support to the idea that flavonoid biosynthesis is normally controlled at the substrate rather than at the enzymic level.

Exogenous substrate utilization by isolated myocytes from chronically diabetic rats.

The effect of chronic streptozotocin-induced diabetes on the utilization of exogenous substrates by freshly isolated, Ca2+-tolerant nonbeating myocytes was investigated. The rates of glucose (5 or 25 mM) and lactate (1 mM) oxidation were significantly reduced in myocytes of diabetic rats, whereas palmitate (0.4 or 1 mM) oxidation was similar to the controls. Glucose oxidation in diabetic (but not in control) and palmitate oxidation in control (but not in diabetic) myocytes were increased by raising the respective substrate concentrations in the medium to levels found in vivo in diabetic rats. Inhibition of glucose and lactate oxidation in the presence of competing substrates were generally similar between control and diabetic myocytes. However, the inhibitory effect of glucose on lactate oxidation was greater in control cells. The rate of palmitate oxidation was diminished by glucose in the controls, but this was not observed in the diabetic myocytes. Oxygen consumption by the myocytes of diabetic rats was below that of control cells when lactate or palmitate was present in the medium. ATP and phosphocreatine contents were similar in the myocytes of diabetic and control rats. All the observed changes in myocytes prepared from diabetic rats were reversed by in vivo insulin treatment.

Exogenous substrate utilization in Ca2+-tolerant myocytes from adult rat hearts.

A preparation of isolated noncontracting adult rat heart myocytes can be obtained in which viability is high, yield is adequate, and the cells are not sensitive to the deleterious effects of Ca2+ at physiological levels. Myocytes are enzymatically dispersed and maintained in a medium containing amino acids and 1% bovine serum albumin at 37 degrees C. The initial viability of 77% fell to 70% after a 60-min incubation in the presence of 1.5 mM Ca2+. The initial cellular ATP content of the myocytes was 27 nmol/mg protein. Cellular ATP content fell to 21 and 17 nmol/mg protein when cells were incubated for 60 min with and without 1.5 mM Ca2+, respectively. The oxidation of glucose and lactate was increased by 50 and 30%, respectively, by the addition of Ca2+. A decrease in the oxidation rate of these two substrates was noted when the myocytes were incubated with medium containing ethyleneglycol-bis(beta-aminoethylether)-N,N'-tetraacetic acid. No changes in the rate of palmitate oxidation were observed. The significant increase in glucose and lactate oxidation due to ca2+ provides further evidence that ca2+ influences myocardial substrate utilization independent of its effects on mechanical performance of the myocardium.

Respiration rates and sugar utilization by marsupial spermatozoa

AbstractThis paper reports the first metabolic study of marsupial spermatozoa. The oxidative metabolism of the spermatozoa of the Australian brush‐tailed possum (Trichosurus vulpecula) was examined using a micro Warburg system. Semen was collected by electro‐ejaculation and washed twice in Ca2+ free Krebs‐Ringer‐phosphate buffer containing antibiotics (KRPA). Washed spermatozoa suspended in fresh KRPA, were then incubated for 3 hours at 37° C in the presence and absence of added substrates (4 mM). The exogenous substrates tested were N‐acetylglucosamine, glucosamine, and glucose. Small quantities of radioactively labeled [14C] substrates were included in the incubation media to allow measurement of substrate oxidation.Although the respiratory rate varied considerably between semen pools (replicate experiments), the relationship between total oxygen consumption (measured manometrically), and oxygen consumption accounted for by exogenous substrate utilization (calcuated from radioactivity recovered in the respiratory CO2) was remarkably consistent. Oxidation of exogenous substrate accounted for 49–54% of the oxygen consumption, depending on the substrate used. There was, however, no evidence that addition of these substrates stimulated the respiratory rate over that found when no substrate was added. Lactate formation accounted for the greater part of exogenous substrate consumed.

Metabolic activity of purified suspensions of Mycobacterium lepraemurium.

SUMMARY: Metabolism of Mycobacterium lepraemurium harvested from infected mice was studied using radioisotope tracer techniques. 14C-labelled acetate, fumarate, glycerol, α-ketoglutarate, glutamine and succinate were oxidized and assimilated, but at extremely low rates. Asparagine and glucose were assimilated but not oxidized. These data provide unequivocal evidence for the utilization of exogenous substrates by purified suspensions of M. lepraemurium and indicate the existence of a host-independent aerobic metabolism and an operative tricarboxylic acid cycle.

Temperature and metabolism in Leishmania. I. Respiration in L. donovani, L. mexicana, and L. tarentolae

Leptomonads of Leishmania donovani, L. mexicana, and L. tarentolae were grown in blood-agar-Locke's-overlay medium, harvested by centrifugation, and their respiration measured by conventional Warburg techniques from 30 to 39 °C in (1) Locke's Solution without glucose, (2) Locke's with glucose, and (3) Locke's with one-half volume hemolyzed, citrated whole rabbit blood. Both the endogenous and substrate-stimulated respiratory rates of all species rose with temperature but dropped above 35 °C in L. mexicana and L. tarentolae and above 37°C in L. donovani. In addition, the respiratory rate of L. mexicana and L. donovani in dilute blood dropped during the first 2 hours of measurement but could be significantly increased by the addition of fresh blood up to 33 °C in L. mexicana and up to 39 °C in L. donovani. It was concluded that the metabolism of the cutaneous L. mexicana was adversely affected at a lower temperature than that of the visceral L. donovani. The respiratory temperature coefficients of the mammalian L. mexicana and L. donovani also differed significantly from those of L. tarentolae (of reptilian origin), indicating perhaps a temperature-stabile system for utilization of exogenous substrate in the latter species.