Metabolomic analyses reveal that anti-aging metabolites are depleted by palmitate but increased by oleate in vivo

Recently, we reported that saturated and unsaturated fatty acids trigger autophagy through distinct signal transduction pathways. Saturated fatty acids like palmitate (PA) induce autophagic responses that rely on phosphatidylinositol 3-kinase, catalytic subunit type 3 (PIK3C3, best known as VPS34) and beclin 1 (BECN1). Conversely, unsaturated fatty acids like oleate (OL) promote non-canonical, PIK3C3- and BECN1-independent autophagy. Here, we explored the metabolic effects of autophagy-inducing doses of PA and OL in mice. Mass spectrometry coupled to principal component analysis revealed that PA and OL induce well distinguishable changes in circulating metabolites as well as in the metabolic profile of the liver, heart, and skeletal muscle. Importantly, PA (but not OL) causes the depletion of multiple autophagy-inhibitory amino acids in the liver. Conversely, OL (but not PA) increased the hepatic levels of nicotinamide adenine dinucleotide (NAD), an obligate co-factor for autophagy-stimulatory enzymes of the sirtuin family. Moreover, PA (but not OL) raised the concentrations of acyl-carnitines in the heart, a phenomenon that perhaps is linked to its cardiotoxicity. PA also depleted the liver from spermine and spermidine, 2 polyamines have been ascribed with lifespan-extending activity. The metabolic changes imposed by unsaturated and saturated fatty acids may contribute to their health-promoting and health-deteriorating effects, respectively.     

Hydrogen peroxide causes greater oxidation in cellular RNA than in DNA

Human A549 lung epithelial cells were challenged with 18O-labeled hydrogen peroxide ([18O]-H2O2), the total RNA and DNA extracted in parallel, and analyzed for 18O-labeled 8-oxo-7,8-dihydroguanosine ([18O]-8-oxoGuo) and 8-oxo-7,8-dihydro-2'-deoxyguanosine ([18O]-8-oxodGuo) respectively, using high-performance liquid chromatography electrospray ionization tandem mass spectrometry (HPLC-MS/MS). [18O]-H2O2 exposure resulted in dose-response formation of both [18O]-8-oxoGuo and [18O]-8-oxodGuo and 18O-labeling of guanine in RNA was 14-25 times more common than in DNA. Kinetics of formation and subsequent removal of oxidized nucleic acids adducts were also monitored up to 24 h. The A549 showed slow turnover rates of adducts in RNA and DNA giving half-lives of approximately 12.5 h for [18O]-8-oxoGuo in RNA and 20.7 h for [18O]-8-oxodGuo in DNA, respectively.     

Reciprocal effects of energy utilization on palmitate oxidation and esterification in hepatocytes of fed rats

The effects of the energy-dependent process of urea synthesis from NH4Cl on the partition of [1-14C]palmitate between oxidation and esterification were examined in hepatocytes of fed rats. A high rate of urea formation from NH4Cl resulted in stimulation of total palmitate oxidation by 25 and 15% at 0.2 and 1 mM fatty acid, respectively. The stimulation of palmitate oxidation was reciprocally correlated with diminished palmitate incorporation into lipids, mainly triacylglycerols. This relationship was almost stoichiometric. NH4Cl increased the palmitate oxidation/esterification ratio from 0.72 to 1.13 and from 0.94 to 1.36 in the presence of 0.2 mM and 1 mM palmitate, respectively. The transaminase inhibitor, aminooxyacetate, strongly inhibited urea synthesis from NH4Cl, had little effect on the low beta-hydroxybutyrate/acetoacetate ratio in the presence of NH4Cl, completely reversed the changes in palmitate metabolism caused by NH4Cl and did not affect palmitate metabolism in the absence of NH4Cl. Therefore, the increased utilization of energy for urea synthesis was the causative factor by which NH4Cl stimulated total palmitate oxidation and led in consequence to its decreased esterification into lipids. Accordingly, these observations indicate that in liver cells the rate of ATP utilization is one of the determinants of triacylglycerol synthesis.     

Compensated cardiac hypertrophy is characterised by a decline in palmitate oxidation

Cardiac hypertrophy is an independent risk factor in the development of heart failure. However, the cellular mechanisms underlying the transition from compensated hypertrophy to heart failure are incompletely understood. The aim of this study was to investigate changes in myocardial substrate utilisation and function in pressure-overload hypertrophy (using ¹³C NMR spectroscopy) in parallel with alterations in the expression pattern of genes involved in cardiac fatty acid and glucose uptake and oxidation. Left ventricular hypertrophy was induced surgically in Sprague–Dawley rats by inter-renal aortic constriction. Nine weeks later, hearts were perfused in the isovolumic mode with a physiological mixture of substrates including 5 mM 1-¹³C glucose, 1 mM 3-¹³C lactate, 0.1 mM U-¹³C pyruvate and 0.3 mM U-¹³C palmitate and cardiac function monitored simultaneously. Real-time PCR was used to determine mRNA levels of PPARα and PPARα-regulated metabolic enzymes. Results showed that at the stage of compensated hypertrophy, fatty acid oxidation (FAO) and expression of genes involved in FAO were markedly reduced, whilst pyruvate oxidation was enhanced, highlighting the fact that metabolic remodelling is an early event in the development of cardiac hypertrophy.     

Arginine synthesis from enteral glutamine in healthy adults in the fed state

Recent studies have documented transfer of labeled nitrogen from [2-(15)N]glutamine to citrulline and arginine in fasting human adults. Conversely, in neonates and piglets we have shown no synthesis of arginine from [2-(15)N]glutamate, and others have shown in mice that glutamine is a nitrogen, but not a carbon donor, for arginine synthesis. Therefore, we performed a multitracer study to determine whether glutamine is a nitrogen and/or carbon donor for arginine in healthy adult men. Two glutamine tracers, 2-(15)N and 1-(13)C, were given enterally to five healthy men fed a standardized milkshake diet. There was no difference in plasma enrichments between the two glutamine tracers. 1-(13)C isotopomers of citrulline and arginine were synthesized from [1-(13)C]glutamine. Three isotopomers each of citrulline and arginine were synthesized from the [2-(15)N]glutamine tracer: 2-(15)N, 5-(15)N, and 2,5-(15)N(2). Significantly greater enrichment was found of both [5-(15)N]arginine (0.75%) and citrulline (3.98%) compared with [2-(15)N]arginine (0.44%) and [2-(15)N]citrulline (2.62%), indicating the amino NH(2) from glutamine is mostly transferred to arginine and citrulline by transamination. Similarly, the enrichment of the 1-(13)C isotopomers was significantly less than the 2-(15)N isotopomers, suggesting rapid formation of α-ketoglutarate and recycling of the nitrogen label. Our results show that the carbon for 50% of newly synthesized arginine comes from dietary glutamine but that glutamine acts primarily as a nitrogen donor for arginine synthesis. Hence, studies using [2-(15)N]glutamine will overestimate arginine synthesis rates.     

Postprandial leg uptake of triglyceride is greater in women than in men

The postprandial excursion of plasma triglyceride (TG) concentration is greater in men than in women. In this study, the disposition of dietary fat was examined in lean healthy men and women (n = 8/group) in either the overnight-fasted or fed (4.5 h after breakfast) states. A [14C]oleate tracer was incorporated into a test meal, providing 30% of total daily energy requirements. After ingestion of the test meal, measures of arteriovenous differences in TG and 14C across the leg were combined with needle biopsies of skeletal muscle and adipose tissue and respiratory gas collections to define the role of skeletal muscle in the clearance of dietary fat. The postprandial plasma TG and 14C tracer excursions were lower (P = 0.04) in women than in men in the overnight-fasted and fed states. Women, however, had significantly greater limb uptake of total TG compared with men on both the fasted (3,849 +/- 846 vs. 528 +/- 221 total micro mol over 6 h) and fed (4,847 +/- 979 vs. 1,571 +/- 334 total micromol over 6 h) days. This was also true for meal-derived 14C lipid uptake. 14C content of skeletal muscle tissue (micro Ci/g tissue) was significantly greater in women than in men 6 h after ingestion of the test meal. In contrast, 14C content of adipose tissue was not significantly different between men and women at 6 h. The main effect of nutritional state, fed vs. fasted, was to increase the postmeal glucose (P = 0.01) excursion (increase from baseline) and decrease the postmeal TG excursion (P = 0.02). These results support the notion that enhanced skeletal muscle clearance of lipoprotein TG in women contributes to their reduced postprandial TG excursion. Questions remain as to the mechanisms causing these sex-based differences in skeletal muscle TG uptake and metabolism. Furthermore, nutritional state can significantly impact postprandial metabolism in both men and women.     

Is insect defoliation in eucalypt forests greater than that in other temperate forests?

Claims have been made in the past that insect defoliation in eucalypt forests of Australia is both chronically high and greater than defoliation levels in northern temperate forests. Recent published data, however, indicate that some eucalypt forests may sustain low level defoliation. Careful reexamination of the literature on defoliation in eucalypt forests indicates that there are not enough data available to quantify defoliation on an Australian-wide basis. Therefore it cannot be claimed that insect defoliation in eucalypt forests is greater than that in other temperate forests. There are also insufficient data to claim that insects are a major factor responsible for the growth differential between indigenous and overseas plantation-grown eucalypts. The ecological impact of herbivorous insects in eucalypt and northern temperate forests is also discussed.     

Influence of malonyl-CoA and palmitate concentration on rate of palmitate oxidation in rat muscle

5-Aminoimidazole-4-carboxamide1--D-ribofuranoside(AICAR) is taken up by perfused skeletal muscle andphosphorylated to form5-aminoimidazole-4-carboxamide-1--D-ribofuraosyl-5'-monophosphate (analog of 5'-AMP) with consequent activation of AMP-activated protein kinase, phosphorylation of acetyl-CoA carboxylase, decrease inmalonyl-CoA, and increase in fatty acid oxidation. Thisstudy was designed to determine the effect of increasing levels ofpalmitate on the rate of fatty acid oxidation. Malonyl-CoAconcentration was manipulated with AICAR at different palmitateconcentrations. Rat hindlimbs were perfused with Krebs-Henseleitbicarbonate containing 4% bovine serum albumin, washed bovine redcells, 200 µU/ml insulin, 10 mM glucose, and different concentrationsof palmitate (0.1-1.0 mM) without or with AICAR (2.0 mM).Perfusion with medium containing AICAR was found to activateAMP-activated protein kinase in skeletal muscle, inactivate acetyl-CoAcarboxylase, and decrease malonyl-CoA at all concentrations ofpalmitate. The rate of palmitate oxidation increased as a function ofpalmitate concentration in both the presence and absence of AICAR butwas always higher in the presence of AICAR. These results provideadditional evidence that malonyl-CoA is an important regulator of therate of fatty acid oxidation at palmitate concentrations in thephysiological range.

     

Unsaturation of mitochondrial membrane lipids is related to palmitate oxidation in subsarcolemmal and intermyofibrillar mitochondria

Membrane lipid composition is thought to influence the function of integral membrane proteins; however, the potential for lipid composition to influence overall mitochondrial long-chain fatty acids (LCFA) oxidation is currently unknown. Therefore, the naturally occurring variability of LCFA oxidation rates within subsarcolemmal (SS) and intermyofibrillar (IMF) mitochondria in muscles with varying oxidative potentials (heart → red → white) was utilized to examine this relationship. To this end, SS and IMF mitochondria were isolated and palmitate oxidation rates were compared to membrane phospholipid composition. Among tissues, rates of palmitate oxidation in mitochondria displayed a 2.5-fold range, creating the required range to determine potential relationships with membrane lipid composition. In general, the percent mole fraction of phospholipid head groups and major fatty acid subclasses were similar in all mitochondria studied. However, rates of palmitate oxidation were positively correlated with both the unsaturation index and relative abundance of cardiolipin within mitochondria (r = 0.57 and 0.49, respectively; p < 0.05). Thus, these results suggest that mitochondrial LCFA oxidation may be significantly influenced by the total unsaturation and percent mole fraction of cardiolipin of the mitochondrial membrane, whereas other indices of membrane structure (e.g., percent mole fraction of other predominant membrane phospholipids, chain length, and ratio of phosphatidylcholine to phosphatidylethanolamine) were not significantly correlated.     

Mechanical strength of the cement-bone interface is greater in shear than in tension.

The objective of this study was to determine the relative mechanical properties of the cement-bone interface due to tensile or shear loading. Mechanical tests were performed on cement-bone specimens in tensile (n = 51) or shear (n = 55) test jigs under the displacement control at 1 mm/min until complete failure. Before testing, the quantity of bone interdigitated with the cement was determined and served as a covariate in the study. The apparent strength of the cement-bone interface was significantly higher (p < 0.0001) for the interface when loaded in shear (2.25 MPa) when compared to tensile loading (1.35 MPa). Significantly higher energies to failure (p < 0.0001) and displacement before failure (p < 0.01) were also determined for the shear specimens. The post-yield softening response was not different for the two test directions. The data obtained herein suggests that cement-bone interfaces with equal amounts of tensile and shear stress would be more likely to fail under tensile loading.     

Molecular expression of myostatin and MyoD is greater in double-muscled than normal-muscled cattle fetuses

Excessive muscling in double-muscled cattle arises from mutations in the myostatin gene, but the role of myostatin in normal muscle development is unclear. The aim of this study was to measure the temporal relationship of myostatin and myogenic regulatory factors during muscle development in normal (NM)- and double-muscled (DM) cattle to determine the timing and possible targets of myostatin action in vivo. Myostatin mRNA peaked at the onset of secondary fiber formation (P < 0.001) and was greater in DM (P < 0.001) than in NM. MyoD expression was also elevated throughout primary and secondary fiber formation (P < 0.001) and greater in DM (P < 0.05). Expression of myogenin peaked later than MyoD (P < 0.05); however, it did not differ between NM and DM. These data show that myostatin and MyoD increase coincidentally during formation of muscle fibers, indicating a coordinated role in the terminal differentiation and/or fusion of myoblasts. Myostatin mRNA is also consistently higher in DM than NM, suggesting that a feedback loop of regulation is also disrupted in the myostatin-deficient condition.     

Strains and stresses in sub-dermal tissues of the buttocks are greater in paraplegics than in healthy during sitting

A pressure-related deep tissue injury (DTI) is a severe pressure ulcer, which initiates in muscle tissue overlying a bony prominence (e.g. the ischial tuberosities, IT) and progresses outwards through fat and skin, unnoticed by the paralyzed patient. We recently showed that internal strains and stresses in muscle and fat of individuals at anatomical sites susceptible to DTI can be evaluated by integrating Open-MRI scans with subject-specific finite element (FE) analyzes (Linder-Ganz et al., Journal of Biomechanics, 2007); however, sub-dermal soft tissue strains/stresses from paraplegics are still missing in literature. We hypothesize that the pathoanatomy of the buttocks in paraplegia increases the internal soft tissue loads under the IT, making these patients inherently susceptible to DTI. We hence compared the strain and stress peaks in the gluteus muscle and fat tissues under the IT of six healthy and six paraplegic patients, using the coupled MRI-FE method. Peak principal compression, principal tension, von Mises and shear strains in the gluteus were 1.2-, 3.1-, 1.4- and 1.4-fold higher in paraplegics than in healthy, respectively (p<0.02). Likewise, peak principal compression, principal tension, von Mises and shear stresses in the gluteus were 1.9-, 2.5-, 2.1- and 1.7-fold higher for the paraplegics (p<0.05). Peak gluteal compression and shear stresses decreased by as much as 70% when the paraplegic patients moved from a sitting to a lying posture, indicating on the effectiveness of recommending such patients to lie down after prolonged periods of sitting. This is the first attempt to compare internal soft tissue loads between paraplegic and healthy subjects, using an objective standardized bioengineering method of analysis. The findings support our hypothesis that internal tissue loads are significantly higher in paraplegics, and that postural changes significantly affect these loads. The method of analysis is useful for quantifying the effectiveness of various interventions to alleviate sub-dermal tissue loads at sites susceptible to pressure ulcers and DTI, including cushions, mattresses, recommendations for posture and postural changes, etc.     

Effects of oleate,palmitate, and octanoate on gluconeogenesis in isolated rabbit liver cells

The effect of oleate, palmitate, and octanoate on glucose formation was studied with lactate or pyruvate as substrate. Octanoate was much more quickly oxidized and utilized for ketone body production than were oleate and palmitate. Among fatty acids studied, only octanoate resulted in a marked increase of the 3-hydroxybutyrate/acetoacetate (3-$\text{OHB}\text{AcAc}$) ratio. Each of the fatty acids studied stimulated glucose synthesis from pyruvate. The enhancement of gluconeogenesis by long-chain fatty acids was abolished after the addition of ammonia. As concluded from the “crossover” plot, the stimulatory effect of fatty acids was due to: (i) a stimulation of pyruvate carboxylation, (ii) a provision of reducing equivalents for glyceraldehyde phosphate dehydrogenase, and (iii) an acceleration of flux through hexose diphosphatase. Moreover, palmitate and oleate resulted in an increased generation of mitochondrial phosphpenolpyruvate, while in the presence of octanoate, the activity of mitochondrial phosphoenolpyruvate carboxykinase was diminished. When lactate was used as the glucose precursor, palmitate and oleate increased glucose production by about 50% but did not affect the contribution of mitochondrial phosphoenolpyruvate carboxykinase to gluconeogenesis. In contrast, in spite of the stimulation of both pyruvate carboxylase and hexose diphosphatase, as judged from the crossover plot, the addition of octanoate resulted in a marked inhibition of both glucose formation and mitochondrial generation of phosphoenolpyruvate. The inhibitory effect of octanoate was reversed by ammonia. Results indicate that fatty acids and ammonia are potent regulatory factors of both the rate of glucose formation and the contribution of mitochondrial phosphoenolpyruvate carboxykinase to gluconeogenesis in hepatocytes of the fasted rabbit.     

Human immunodeficiency virus type 1 genetic recombination is more frequent than that of Moloney murine leukemia virus despite similar template switching rates

Retroviral recombinants result from template switching between copackaged viral genomes. Here, marker reassortment between coexpressed vectors was measured during single replication cycles, and human immunodeficiency virus type 1 (HIV-1) recombination was observed six- to sevenfold more frequently than murine leukemia virus (MLV) recombination. Template switching was also assayed by using transduction-type vectors in which donor and acceptor template regions were joined covalently. In this situation, where RNA copackaging could not vary, MLV and HIV-1 template switching rates were indistinguishable. These findings argue that MLV's lower intermolecular recombination frequency does not reflect enzymological differences. Instead, these data suggest that recombination rates differ because coexpressed MLV RNAs are less accessible to the recombination machinery than are coexpressed HIV RNAs. This hypothesis provides a plausible explanation for why most gammaretrovirus recombinants, although relatively rare, display evidence of multiple nonselected crossovers. By implying that recombinogenic template switching occurs roughly four times on average during the synthesis of every MLV or HIV-1 DNA, these results suggest that virtually all products of retroviral replication are biochemical recombinants.     

The oxidation and utilization of palmitate, stearate, oleate and acetate by the mammary gland of the fed goat in relation to their overall metabolism, and the role of plasma phospholipids and neutral lipids in milk-fat synthesis

1. Measurements were made of milk yield, mammary blood flow and arteriovenous differences of each plasma lipid fraction, and their specific radioactivities, during the infusion of [U-¹⁴C]stearate, [U-¹⁴C]oleate, [U-¹⁴C]palmitate and [1-¹⁴C]acetate into fed lactating goats. 2. Entry rates of fatty acids into the circulation were 4·2mg./min./kg. body wt. for acetate, and 0·18, 0·28 and 0·42mg./min./kg. for stearate, oleate and palmitate respectively. Acetate accounted for 23% of the total carbon dioxide produced by the whole animal, and contributed to the oxidative metabolism of the mammary gland to about the same extent. Corresponding values for each of the long-chain acids were less than 1%. 3. There were no significant arteriovenous differences of phospholipids, sterols or sterol esters, and their fatty acid composition showed no net changes during passage through the mammary gland. 4. There were large arteriovenous differences of plasma triglycerides, and their fatty acid composition showed marked changes across the gland. The proportions of palmitate and stearate fell, and that of oleate increased. 5. Arteriovenous differences of plasma free fatty acids (FFA) were small and variable, but a large fall in the specific radioactivity of each of the long-chain acids examined indicated substantial uptake of plasma FFA, accompanied by roughly equivalent FFA release from mammary tissue. The uptake of FFA was confirmed by the extensive transfer of radioactivity into milk. The FFA of milk were similar in composition and radioactivity to the milk triglyceride fatty acids, and quite unlike plasma FFA. 6. The formation of large amounts of oleic acid (18–21 mg./min.) from stearic acid was demonstrated. 7. During the terminal stages of the [¹⁴C]acetate infusion, milk triglyceride fatty acids of chain length C₄–C₁₄ showed specific radioactivities that were 75–90% of that of blood acetate, and that of palmitate was roughly one-quarter of this value. Oleate and stearate were unlabelled. 8. The results confirmed that milk fatty acids of chain length C₄–C₁₄ arise largely from blood acetate, and palmitate is derived partly from acetate and partly from plasma triglyceride, the latter fraction being almost the sole precursor of oleate and stearate.