Quantifying Sphingomyelin in Dairy through Infusion-Based Shotgun Mass Spectrometry with Lithium-Ion-Induced Fragmentation.

Quantifying sphingomyelin (SM) species by infusion-based mass spectrometry (MS) is complicated by the presence of isobaric phosphatidylcholine (PC) species, which generate a common m/z 184 product ion in the presence of ammonium ions as a result of the phosphocholine headgroup. Lithium ion adducts of SM undergo a selective dehydration [Li + H2O + (CH3)3NC2H4PO4] with a corresponding neutral loss of -207 Da. This neutral loss was employed to create a SM-selective method for identifying target species, which were quantitated using multiple reaction monitoring (MRM). SM-selective fragments in MS3 were used to characterize the sphingosine base and acyl chain. These methods were used to identify 50 individual SM species in bovine milk ranging from SM 28:1 to SM 44:2, with d16:1, d17:1, d18:1, d19:1, and d20:1 bases, and acyl fatty acids ranging from 10 to 25 carbons and 0-1 desaturations. Spiked SM standards into milk had a recovery of 99.7%, and endogenous milk SM had <10% coefficient of variation for both intra- and interday variability, with limits of detection of 1.4-5.55 nM and limits of quantitation of 11.8-178.1 nM. This MS-MRM method was employed to accurately and precisely quantify SM species in dairy products, including bovine-derived whole milk, half and half, whipping cream, and goat milk.

Lesch-Nyhan disease causes impaired energy metabolism and reduced developmental potential in midbrain dopaminergic cells.

Mutations in HPRT1, a gene encoding a rate-limiting enzyme for purine salvage, cause Lesch-Nyhan disease which is characterized by self-injury and motor impairments. We leveraged stem cell and genetic engineering technologies to model the disease in isogenic and patient-derived forebrain and midbrain cell types. Dopaminergic progenitor cells deficient in HPRT showed decreased intensity of all developmental cell-fate markers measured. Metabolic analyses revealed significant loss of all purine derivatives, except hypoxanthine, and impaired glycolysis and oxidative phosphorylation. real-time glucose tracing demonstrated increased shunting to the pentose phosphate pathway for de novo purine synthesis at the expense of ATP production. Purine depletion in dopaminergic progenitor cells resulted in loss of RHEB, impairing mTORC1 activation. These data demonstrate dopaminergic-specific effects of purine salvage deficiency and unexpectedly reveal that dopaminergic progenitor cells are programmed to a high-energy state prior to higher energy demands of terminally differentiated cells.

Ca2+ entry via TRPC1 is essential for cellular differentiation and modulates secretion via the SNARE complex.

Properties of adipocytes, including differentiation and adipokine secretion, are crucial factors in obesity-associated metabolic syndrome. Here, we provide evidence that Ca2+ influx in primary adipocytes, especially upon Ca2+ store depletion, plays an important role in adipocyte differentiation, functionality and subsequently metabolic regulation. The endogenous Ca2+ entry channel in both subcutaneous and visceral adipocytes was found to be dependent on TRPC1-STIM1, and blocking Ca2+ entry with SKF96365 or using TRPC1-/- knockdown adipocytes inhibited adipocyte differentiation. Additionally, TRPC1-/- mice have decreased organ weight, but increased adipose deposition and reduced serum adiponectin and leptin concentrations, without affecting total adipokine expression. Mechanistically, TRPC1-mediated Ca2+ entry regulated SNARE complex formation, and agonist-mediated secretion of adipokine-loaded vesicles was inhibited in TRPC1-/- adipose. These results suggest an unequivocal role of TRPC1 in adipocyte differentiation and adiponectin secretion, and that loss of TRPC1 disturbs metabolic homeostasis.This article has an associated First Person interview with the first author of the paper.

Increasing acetyl-CoA metabolism attenuates injury and alters spinal cord lipid content in mice subjected to experimental autoimmune encephalomyelitis.

Acetate supplementation increases brain acetyl-CoA metabolism, alters histone and non-histone protein acetylation, increases brain energy reserves, and is anti-inflammatory and neuroprotective in rat models of neuroinflammation and neuroborreliosis. To determine the impact acetate supplementation has on a mouse model of multiple sclerosis, we quantified the effect treatment had on injury progression, spinal cord lipid content, phospholipase levels, and myelin structure in mice subjected to experimental autoimmune encephalomyelitis (EAE). EAE was induced by inoculating mice with a myelin oligodendrocyte glycoprotein peptide fragment (MOG35-55 ), and acetate supplementation was maintained with 4 g/kg glyceryl triacetate by a daily oral gavage. Acetate supplementation prevented the onset of clinical signs in mice subject to EAE compared to control-treated mice. Furthermore, acetate supplementation prevented the loss of spinal cord ethanolamine and choline glycerophospholipid and phosphatidylserine in mice subjected to EAE compared to EAE animals treated with water. Treatment increased saturated and monounsaturated fatty acid levels in phosphatidylserine compared to controls suggesting that acetate was utilized to increase spinal cord fatty acid content. Also, acetate supplementation prevented the loss of spinal cord cholesterol in EAE animals but did not change cholesteryl esters. Treatment significantly increased GD3 and GD1a ganglioside levels in EAE mice when compared to EAE mice treated with water. Treatment returned levels of phosphorylated and non-phosphorylated cytosolic phospholipase A2 (cPLA2 ) levels back to baseline and based on FluoroMyelin™ histochemistry maintained myelin structural characteristics. Overall, these data suggest that acetate supplementation may modulate lipid metabolism in mice subjected to EAE.

Quantitation of isobaric phosphatidylcholine species in human plasma using a hybrid quadrupole linear ion-trap mass spectrometer.

Phosphatidylcholine (PC) species in human plasma are used as biomarkers of disease. PC biomarkers are often limited by the inability to separate isobaric PCs. In this work, we developed a targeted shotgun approach for analysis of isobaric and isomeric PCs. This approach is comprised of two MS methods: a precursor ion scanning (PIS) of mass m/z 184 in positive mode (PIS m/z +184) and MS3 fragmentation in negative mode, both performed on the same instrument, a hybrid triple quadrupole ion-trap mass spectrometer. The MS3 experiment identified the FA composition and the relative abundance of isobaric and sn-1, sn-2 positional isomeric PC species, which were subsequently combined with absolute quantitative data obtained by PIS m/z +184 scan. This approach was applied to the analysis of a National Institute of Standards and Technology human blood plasma standard reference material (SRM 1950). We quantified more than 70 PCs and confirmed that a majority are present in isobaric and isomeric mixtures. The FA content determined by this method was comparable to that obtained using GC with flame ionization detection, supporting the quantitative nature of this MS method. This methodology will provide more in-depth biomarker information for clinical and mechanistic studies.

Twice weekly intake of farmed Atlantic salmon (Salmo salar) positively influences lipoprotein concentration and particle size in overweight men and women.

The US Dietary Guidelines for Americans recommend twice weekly fish intake. Farmed Atlantic salmon is a good source of omega-3 (n-3) fatty acids which have positive lipid modifying effects; however, it is unknown whether these responses are dose-dependent. Our primary research objective was to determine the effect of dose-dependent intake of farmed Atlantic salmon on lipoprotein particle (P) size and concentration. We hypothesized that low-density lipoprotein (LDL)-P and high-density lipoprotein (HDL)-P size and concentration would increase with salmon intake in a dose-dependent manner. Overweight, adult participants (n = 19) were enrolled in a cross-over designed clinical trial evaluating intake of farmed Atlantic salmon. In random order, participants were assigned to 90, 180, or 270 g of salmon twice weekly for 4-week dietary treatments. Following a 4- to 8-week washout, participants crossed over to another dose of fish intake until all treatments were completed. Plasma lipid concentrations were determined and serum lipoprotein concentrations and particle size were determined by nuclear magnetic resonance. Intake of salmon reduced plasma and serum triglyceride (TG) concentrations and increased plasma HDL-C concentrations. The concentrations of large very low-density lipoprotein (VLDL)-P and chylomicron (CM)-P were reduced. Large LDL-P concentrations were increased in a dose-dependent manner. The mean size of VLDL-P was reduced and that of LDL was increased. Total TG was reduced as was the TG content of VLDL-P and CM-P. Twice weekly intake of farmed Atlantic salmon portions influences lipoprotein particle size and concentration in a manner associated with cardiovascular disease risk reduction.

Acetate supplementation modulates brain adenosine metabolizing enzymes and adenosine A2A receptor levels in rats subjected to neuroinflammation.

BACKGROUND: Acetate supplementation reduces neuroglia activation and pro-inflammatory cytokine expression in rat models of neuroinflammation and Lyme neuroborreliosis. Because single-dose glyceryl triacetate (GTA) treatment increases brain phosphocreatine and reduces brain AMP levels, we postulate that GTA modulates adenosine metabolizing enzymes and receptors, which may be a possible mechanism to reduce neuroinflammation. METHODS: To test this hypothesis, we quantified the ability of GTA to alter brain levels of ecto-5'-nucleotidase (CD73), adenosine kinase (AK), and adenosine A2A receptor using western blot analysis and CD73 activity by measuring the rate of AMP hydrolysis. Neuroinflammation was induced by continuous bacterial lipopolysaccharide (LPS) infusion in the fourth ventricle of the brain for 14 and 28 days. Three treatment strategies were employed, one and two where rats received prophylactic GTA through oral gavage with LPS infusion for 14 or 28 days. In the third treatment regimen, an interventional strategy was used where rats were subjected to 28 days of neuroinflammation, and GTA treatment was started on day 14 following the start of the LPS infusion. RESULTS: We found that rats subjected to neuroinflammation for 28 days had a 28% reduction in CD73 levels and a 43% increase in AK levels that was reversed with prophylactic acetate supplementation. CD73 activity in these rats was increased by 46% with the 28-day GTA treatment compared to the water-treated rats. Rats subjected to neuroinflammation for 14 days showed a 50% increase in levels of the adenosine A2A receptor, which was prevented with prophylactic acetate supplementation. Interventional GTA therapy, beginning on day 14 following the induction of neuroinflammation, resulted in a 67% increase in CD73 levels and a 155% increase in adenosine A2A receptor levels. CONCLUSION: These results support the hypothesis that acetate supplementation can modulate brain CD73, AK and adenosine A2A receptor levels, and possibly influence purinergic signaling.

Polyunsaturated fatty acid content may be increased in the milk of women with pregnancy-associated breast cancer.

BACKGROUND: Pregnancy-associated breast cancer (PABC) is aggressive and difficult to diagnose. High intake of most types of dietary fat is thought to increase breast cancer risk; however, results in humans supporting this premise remain equivocal. Fatty acid (FA) concentrations in the body comprise both dietary intake and endogenous FA production. Most assessments of FA levels have been performed on blood, with little information on the effect of FA levels in breast milk on PABC risk. OBJECTIVE: This study aimed to determine if FA concentrations in the milk from women diagnosed with breast cancer while nursing were different in the cancer-containing breast and opposite breast. METHODS: We quantified 16 long-chain FA and soluble FA synthase (sFAS) enzyme levels from 4 women diagnosed with PABC, comparing results from the cancer-containing breast to those from the normal breast. RESULTS: Fatty acid concentrations consistently exceeded and trended higher (P < .10) in each cancer-containing breast for 20:4n-6 (arachidonic acid [AA]), 20:5n-3 (eicosapentaenoic acid [EPA]), and 22:5n-6 (docosapentaenoic acid [DPA]). Soluble FA synthase levels were similar in the cancer-containing and normal breasts. CONCLUSION: Breast milk concentrations of AA, EPA, and DPA increased in the cancer-containing breast of women with PABC. This increase was not associated with higher sFAS levels.

Acetate treatment increases fatty acid content in LPS-stimulated BV2 microglia.

Acetate supplementation increases plasma acetate, brain acetyl-CoA, histone acetylation, phosphocreatine levels, and is anti-inflammatory in models of neuroinflammation and neuroborreliosis. Although radiolabeled acetate is incorporated into the cellular lipid pools, the effect that acetate supplementation has on lipid deposition has not been quantified. To determine the impact acetate-treatment has on cellular lipid content, we investigated the effect of acetate in the presence of bacterial lipopolysaccharide (LPS) on fatty acid, phospholipid, and cholesterol content in BV2 microglia. We found that 1, 5, and 10 mM of acetate in the presence of LPS increased the total fatty acid content in BV2 cells by 23, 34, and 14 % at 2 h, respectively. Significant increases in individual fatty acids were also observed with all acetate concentrations tested with the greatest increases occurring with 5 mM acetate in the presence of LPS. Treatment with 5 mM acetate in the absence of LPS increased total cholesterol levels by 11 %. However, neither treatment in the absence of LPS significantly altered the content of individual phospholipids or total phospholipid content. To determine the minimum effective concentration of acetate we measured the time- and concentration-dependent changes in histone acetylation using western blot analysis. These studies showed that 5 mM acetate was necessary to induce histone acetylation and at 10 mM acetate, the histone acetylation-state increased as early as 0.5 h following the start of treatment. These data suggest that acetate increases fatty acid content in LPS-stimulated BV2 microglia that is reflected by an increase in fatty acids esterified into membrane phospholipids.

Bacterial lipopolysaccharide induces a dose-dependent activation of neuroglia and loss of basal forebrain cholinergic cells in the rat brain.

In a rat model of neuroinflammation induced with a low-dose infusion lipopolysaccharide (5.0 ng/hr, LPS), we reported that brain arachidonic acid (ARA, 20:4 n-6), but not docosahexaenoic acid (DHA, 22:6n-3), metabolism is increased compared to control rats. To further characterize the impact LPS has on the induction of injury in this model, we quantified the dose-dependent activation of neuroglia and the loss of cholinergic cells in rats subjected to increasing doses of LPS. In this study, we found that LPS produced a statistically significant and linear dose-dependent increase in the percentage of activated CD11b-positive microglia ranging from 26% to 82% following exposure to doses ranging between 0.05 and 500 ng/hr, respectively. The percentage of activated GFAP-positive astrocytes also increased linearly and significantly from 35% to 91%. Significant astroglial scaring was evident at the lateral ventricular boarder of rats treated with 50 and 500 ng/hr LPS, but not evident in control treated rats or rats treated with lower doses of LPS. A dose-dependent decrease in the numbers of ChAT-positive cells in the basal forebrain of LPS-treated rats was found at higher doses of LPS (5, 50, and 500 ng/hr) but not at lower doses. The numbers of ChAT-positive cells within individual regions of the basal forebrain (medial septum and diagonal bands) and the composite basal forebrain were similar in their response. These data demonstrate that extremely low doses of LPS are sufficient to induce significant neuroglia activation while moderate doses above 5.0 ng/hr are required to induce cholinergic cell loss.

Acetate reduces PGE2 release and modulates phospholipase and cyclooxygenase levels in neuroglia stimulated with lipopolysaccharide.

Acetate supplementation attenuates neuroglial activation, increases histone and non-histone protein acetylation, reduces pro-inflammatory cytokine expression, and increases IL-4 transcription in rat models of neuroinflammation and Lyme's neuroborreliosis. Because eicosanoid signaling is involved in neuroinflammation, we measured the effect acetate treatment had on phospholipase, cyclooxygenase, and prostaglandin E2 (PGE2) levels in BV-2 microglia and primary astrocytes stimulated with lipopolysaccharide (LPS). In BV-2 microglia, we found that LPS increased the phosphorylation-state of cytosolic phospholipase A2 (cPLA2), reduced the levels of phospholipase C (PLC) ß1, and increased the levels of cyclooxygenase (Cox)-1 and -2. Acetate treatment returned PLCß1 and Cox-1 levels to normal, attenuated the increase in Cox-2, but had no effect on cPLA2 phosphorylation. In primary astrocytes, LPS increased the phosphorylation of cPLA2 and increased the levels of Cox-1 and Cox-2. Acetate treatment in these cells reduced secretory PLA2 IIA and PLCß1 levels as compared to LPS-treatment groups, reversed the increase in cPLA2 phosphorylation, and returned Cox-1 levels to normal. Acetate treatment reduced PGE2 release in astrocytes stimulated with LPS to control levels, but did not alter PGE2 levels in BV-2 microglia. The amount of acetylated H3K9 bound to the promoter regions of Cox-1, Cox-2, IL-1ß and NF-κB p65 genes, but not IL-4 in were increased in BV-2 microglia treated with acetate. These data suggest that acetate treatment can disrupt eicosanoid signaling in neuroglia that may, in part, be the result of altering gene expression due chromatin remodeling as a result of increasing H3K9 acetylation.

Dose-dependent consumption of farmed Atlantic salmon (Salmo salar) increases plasma phospholipid n-3 fatty acids differentially.

Enhanced n-3 fatty acid intake benefits cardiovascular disease (CVD) risk reduction. Increasing consumption at a population level may be better addressed by diet than through supplementation. However, limited data are available on the effect of the dose response to fish intake on plasma levels of n-3 fatty acids. To compare the effects of different doses of farmed Atlantic salmon on plasma phospholipid fatty acid proportions and CVD risk biomarkers (eg, glucose, insulin, homeostasis model of assessment-insulin resistance, high-sensitivity C-reactive protein, and interleukin-6) in healthy subjects we performed a randomized three-period crossover-designed trial (4-week treatment, 4- to 8-week washout) to compare the effects of twice per week consumption of farmed Atlantic salmon at doses of 90, 180, and 270 g in 19 apparently healthy men and women (mean age 40 to 65 years) and a body mass index between 25 and 34.9. All study visits were conducted at the US Department of Agriculture Agricultural Research Service Grand Forks Human Nutrition Research Center. Eicosapentaenoic acid and total n-3 concentrations were increased (P<0.05) by all treatments in a dose-response manner, with total n-3 of 8.03% ± 0.26% and 9.21% ± 0.26% for 180- and 270-g doses, respectively. Linoleic acid did not change in response to treatment, whereas arachidonic acid (P<0.05) and total n-6 fatty acids decreased dose dependently (<0.0001). The addition of farmed Atlantic salmon to the diet twice per week for 4 weeks at portions of 180 g and 270 g modifies phospholipid fatty acid proportions of n-3 and n-6 in a level associated with decreased risk for CVD.

Acetate supplementation increases brain phosphocreatine and reduces AMP levels with no effect on mitochondrial biogenesis.

Acetate supplementation in rats increases plasma acetate and brain acetyl-CoA levels. Although acetate is used as a marker to study glial energy metabolism, the effect that acetate supplementation has on normal brain energy stores has not been quantified. To determine the effect(s) that an increase in acetyl-CoA levels has on brain energy metabolism, we measured brain nucleotide, phosphagen and glycogen levels, and quantified cardiolipin content and mitochondrial number in rats subjected to acetate supplementation. Acetate supplementation was induced with glyceryl triacetate (GTA) by oral gavage (6 g/kg body weight). Rats used for biochemical analysis were euthanized using head-focused microwave irradiation at 2, and 4h following treatment to immediately stop metabolism. We found that acetate did not alter brain ATP, ADP, NAD, GTP levels, or the energy charge ratio [ECR, (ATP+½ ADP)/(ATP+ADP+AMP)] when compared to controls. However, after 4h of treatment brain phosphocreatine levels were significantly elevated with a concomitant reduction in AMP levels with no change in glycogen levels. In parallel studies where rats were treated with GTA for 28 days, we found that acetate did not alter brain glycogen and mitochondrial biogenesis as determined by measuring brain cardiolipin content, the fatty acid composition of cardiolipin and using quantitative ultra-structural analysis to determine mitochondrial density/unit area of cytoplasm in hippocampal CA3 neurons. Collectively, these data suggest that an increase in brain acetyl-CoA levels by acetate supplementation does increase brain energy stores however it has no effect on brain glycogen and neuronal mitochondrial biogenesis.

Modulation of inflammatory cytokines and mitogen-activated protein kinases by acetate in primary astrocytes.

Acetate supplementation attenuates neuroglia activation in a rat model of neuroinflammation by a mechanism associated with an increase in brain acetyl-CoA, an alteration in histone acetylation, and reduction of interleukin (IL)-1ß expression. We propose that reduced astroglial activation occurs by disrupting astrocyte-derived inflammatory signaling and cytokine release. Using primary astroglial cultures, we found that LPS (0-25 ng/ml, 4 h) increased tumor necrosis factor (TNF-α) and IL-1ß in a concentration-dependent manner, which was reduced by treatment with sodium acetate (12 mM). LPS did not alter H3K9 acetylation or IL-6 levels, whereas acetate treatment increased H3K9 acetylation by 2-fold and decreased basal levels of IL-6 by 2-fold. Acetate treatment attenuated the LPS-induced increase in TNF-α mRNA, but did not reverse the mRNA levels of other pro-inflammatory cytokines. By contrast, LPS decreased TGF-ß1 and IL-4 protein and TGF-ß1 mRNA, all of which was reversed with acetate treatment. Further, we found that acetate treatment completely reversed LPS-induced phosphorylation of MAPK p38 and decreased basal levels of phosphorylated extracellular signal-regulated kinases1/2 (ERK1/2) by 2-fold. Acetate treatment also reversed LPS-elevated NF-κB p65, CCAAT/enhancer-binding protein beta protein levels, and reduced basal levels of phosphorylated NF-κB p65 at serine 536. These results suggest that acetate treatment has a net anti-inflammatory effect in LPS-stimulated astrocytes that is largely associated with a disruption in MAPK and NF-κB signaling.

Acetate supplementation reduces microglia activation and brain interleukin-1ß levels in a rat model of Lyme neuroborreliosis.

BACKGROUND: We have found that acetate supplementation significantly reduces neuroglia activation and pro-inflammatory cytokine release in a rat model of neuroinflammation induced with lipopolysaccharide. To test if the anti-inflammatory effect of acetate supplementation is specific to a TLR4-mediated injury, we measured markers of neuroglia activation in rats subjected to B. burgdorferi-induced neuroborreliosis that is mediated in large part by a TLR2-type mechanism. METHODS: In this study, rats were subjected to Lyme neuroborreliosis following an intravenous infusion of B. burgdorferi (B31-MI-16). Acetate supplementation was induced using glyceryl triacetate (6g/kg) by oral gavage. Immunohistochemistry, qPCR, and western blot analyses were used to measure bacterial invasion into the brain, neuroglial activation, and brain and circulating levels of interleukin 1ß. Statistical analysis was performed using one-way analysis of variance (ANOVA) followed by a Tukey's post hoc tests or using a Student's t test assuming unequal variances when appropriate. RESULTS: We found that acetate supplementation significantly reduced microglia activation by 2-fold as determined by immunohistochemical and western blot analysis. Further, acetate supplementation also reduced the expression of the pro-inflammatory cytokine IL-1ß by 2-fold as compared to controls. On the other hand, the inoculation of rats with B. burgdorferi had no effect on astroglial activation as determined by immunocytochemistry and western blot analysis despite significant increases in circulation levels of antigen toward B. burgdorferi and presence of the bacteria in the central nervous system. CONCLUSIONS: These results suggest that microglial activation is an essential component to neuroborreliosis and that acetate supplementation may be an effective treatment to reduce injury phenotype and possibly injury progression in Lyme neuroborreliosis.

Acetate reduces microglia inflammatory signaling in vitro.

Acetate supplementation increases brain acetyl-CoA and histone acetylation and reduces lipopolysaccharide (LPS)-induced neuroglial activation and interleukin (IL)-1ß expression in vivo. To determine how acetate imparts these properties, we tested the hypothesis that acetate metabolism reduces inflammatory signaling in microglia. To test this, we measured the effect acetate treatment had on cytokine expression, mitogen-activated protein kinase (MAPK) signaling, histone H3 at lysine 9 acetylation, and alterations of nuclear factor-kappa B (NF-κB) in primary and BV-2 cultured microglia. We found that treatment induced H3K9 hyperacetylation and reversed LPS-induced H3K9 hypoacetylation similar to that found in vivo. LPS also increased IL-1ß, IL-6, and tumor necrosis factor-alpha (TNF-α) mRNA and protein, whereas treatment returned the protein to control levels and only partially attenuated IL-6 mRNA. In contrast, treatment increased mRNA levels of transforming growth factor-ß1 (TGF-ß1) and both IL-4 mRNA and protein. LPS increased p38 MAPK and JNK phosphorylation at 4 and 2-4 h, respectively, whereas treatment reduced p38 MAPK and JNK phosphorylation only at 2 h. In addition, treatment reversed the LPS-induced elevation of NF-κB p65 protein and phosphorylation at serine 468 and induced acetylation at lysine 310. These data suggest that acetate metabolism reduces inflammatory signaling and alters histone and non-histone protein acetylation.

Ketone bodies protection against HIV-1 Tat-induced neurotoxicity.

HIV-1-associated neurocognitive disorder (HAND) is a syndrome that ranges clinically from subtle neuropsychological impairments to profoundly disabling HIV-associated dementia. Not only is the pathogenesis of HAND unclear, but also effective treatments are unavailable. The HIV-1 transactivator of transcription protein (HIV-1 Tat) is strongly implicated in the pathogenesis of HAND, in part, because of its well-characterized ability to directly excite neurons and cause neurotoxicity. Consistent with previous findings from others, we demonstrate here that HIV-1 Tat induced neurotoxicity, increased intracellular calcium, and disrupted a variety of mitochondria functions, such as reducing mitochondrial membrane potential, increasing levels of reactive oxygen species, and decreasing bioenergetic efficiency. Of therapeutic importance, we show that treatment of cultured neurons with ketone bodies normalized HIV-1 Tat induced changes in levels of intracellular calcium, mitochondrial function, and neuronal cell death. Ketone bodies are normally produced in the body and serve as alternative energy substrates in tissues including brain and can cross the blood-brain barrier. Ketogenic strategies have been used clinically for treatment of neurological disorders and our current results suggest that similar strategies may also provide clinical benefits in the treatment of HAND.

Acetate supplementation modulates brain histone acetylation and decreases interleukin-1ß expression in a rat model of neuroinflammation.

BACKGROUND: Long-term acetate supplementation reduces neuroglial activation and cholinergic cell loss in a rat model of lipopolysaccharide-induced neuroinflammation. Additionally, a single dose of glyceryl triacetate, used to induce acetate supplementation, increases histone H3 and H4 acetylation and inhibits histone deacetylase activity and histone deacetylase-2 expression in normal rat brain. Here, we propose that the therapeutic effect of acetate in reducing neuroglial activation is due to a reversal of lipopolysaccharide-induced changes in histone acetylation and pro-inflammatory cytokine expression. METHODS: In this study, we examined the effect of a 28-day-dosing regimen of glyceryl triacetate, to induce acetate supplementation, on brain histone acetylation and interleukin-1ß expression in a rat model of lipopolysaccharide-induced neuroinflammation. The effect was analyzed using Western blot analysis, quantitative real-time polymerase chain reaction and enzymic histone deacetylase and histone acetyltransferase assays. Statistical analysis was performed using one-way analysis of variance, parametric or nonparametric when appropriate, followed by Tukey's or Dunn's post-hoc test, respectively. RESULTS: We found that long-term acetate supplementation increased the proportion of brain histone H3 acetylated at lysine 9 (H3K9), histone H4 acetylated at lysine 8 and histone H4 acetylated at lysine 16. However, unlike a single dose of glyceryl triacetate, long-term treatment increased histone acetyltransferase activity and had no effect on histone deacetylase activity, with variable effects on brain histone deacetylase class I and II expression. In agreement with this hypothesis, neuroinflammation reduced the proportion of brain H3K9 acetylation by 50%, which was effectively reversed with acetate supplementation. Further, in rats subjected to lipopolysaccharide-induced neuroinflammation, the pro-inflammatory cytokine interleukin-1ß protein and mRNA levels were increased by 1.3- and 10-fold, respectively, and acetate supplementation reduced this expression to control levels. CONCLUSION: Based on these results, we conclude that dietary acetate supplementation attenuates neuroglial activation by effectively reducing pro-inflammatory cytokine expression by a mechanism that may involve a distinct site-specific pattern of histone acetylation and histone deacetylase expression in the brain.

A sensitive HPLC-based method to quantify adenine nucleotides in primary astrocyte cell cultures.

In mono-layered primary cell cultures baseline AMP and ADP levels are found nominally in the mid to low picomolar range and are thus difficult to measure with conventional HPLC methods that often require the pooling of samples or require indirect detection methods using radiotracers or enzyme coupled assays. To address this issue, we developed a highly sensitive and selective ion-pairing HPLC method with fluorescence detection to quantify adenine nucleotides and the adenylate energy charge in primary astrocyte cell cultures. To accomplish this, we optimized the fluorescence derivatization conditions and the HPLC parameters to achieve baseline separation and quantification of all adenine nucleotides. Nucleotides were converted to their respective 1, N(6)-etheno derivatives by incubating with chloroacetaldehyde at pH 4.5 and 60°C for 60 min. Under these conditions, the loss of the adenine nucleotides due to hydrolysis was minimized with a derivatization yield of 94.1% for 1, N(6)-ethenoadenosine. The optimal concentration of tetrabutylammonium phosphate, the ion-pairing reagent, required to achieve a reproducible separation of the adenine nucleotides was found to be 0.8mM. Calibration curves of nucleotide standards were linear within the range of 0.16-10.4 pmol for adenosine, 0.16-20.6 pmol for AMP, 0.15-19.2 pmol for ADP, and 0.15-19.5 pmol for ATP. The limits of detection and quantification for all adenine nucleotides were approximately 0.08 and 0.16 pmol, respectively. The intra- and inter-day variability for this method was less than 5.1 and 3.4%, respectively. This method was successfully used to measure all adenine nucleotides and an adenylate energy charge of 0.92±0.02 in primary astrocyte cell cultures.