Retinol

Lipidomics Gateway (28 September 2011) [doi:10.1038/lipidmaps.2011.26]

Retinol and its metabolites mediate a myriad of physiological functions, from vision to reproduction.

The structure of retinol (also known as vitamin A). Visit retinol in the LIPID MAPS structure database for more molecular information.

The prenol lipid retinol (also known as vitamin A) is the immediate precursor to an aldehyde metabolite, retinal, which is required for visual function, and an acid metabolite, retinoic acid, which regulates many other, different, physiological functions. Structurally, retinol and its metabolites possess a β-ionone ring and a polyunsaturated side chain to which an alcohol, aldehyde or carboxylic acid group is attached.

Retinol is obtained directly from foods of animal origin, such as liver and eggs, and indirectly from carotenoids (including α-carotene, β-carotene, γ-carotene or β-cryptoxanthin), which are found in carrots and spinach. Any excess of this fat-soluble vitamin is stored in the liver and fatty tissues as retinyl esters, which can be readily hydrolysed to retinol, when necessary, and transported in the blood bound to retinol-binding protein. However, vitamin A toxicity can occur because, being fat-soluble, it is difficult to eliminate from the body.

Vitamin A deficiency is the main cause of blindness in the developing world, highlighting the key role of retinal in vision. In photoreceptor cells within the retina, 11-cis-retinal binds to different opsin receptors ¹ ; when it absorbs light, it is isomerized to form 11-trans-retinal. This physical change triggers a chain of events that ultimately alters the signals sent along the optic nerve to the visual centre of the brain. In addition to this key role in vision, retinal has recently been reported to inhibit adipogenesis ² .

Retinoic acid mediates an abundance of physiological effects, including immunity, reproduction and embryonic development, primarily through gene transcription. Upon binding its all-trans-retinoic acid ligand, the retinoic acid receptor heterodimerizes with a retinoid X receptor and binds to retinoic acid response elements on DNA to modify the expression of target genes. More recently, a role for retinoic acid in regulating protein translation has also been discovered ³ .

Although its metabolites are thought to be responsible for the majority of effects, retinol itself also has distinct biological activities ¹ . For example, retinol is transported into cells by the STRA6 membrane transporter, which has been shown to induce signal transduction leading to gene transcription in response to association with the retinol–RBP complex ⁴ .

The finding that vitamin A administration can decrease childhood mortality by 20–70% in developing countries underscores the impact of retinol on human health, and has led to schemes aimed at enriching staple foods with vitamin A ¹ . Similarly, retinoid therapy is advocated for several diseases, including cancer, metabolic diseases and dermatological diseases.

Katrin Legg

References:

1. Sun, H. Membrane receptors and transporters involved in the function and transport of vitamin A and its derivatives.
   Biochim. Biophys. Acta (2011). doi:10.1016/j.bbalip.2011.06.010
2. Ziouzenkova, O. et al. Retinaldehyde represses adipogenesis and diet-induced obesity.
   Nat. Med. 13, 695-702 (2007). doi:10.1038/nm1587
3. Chen, N., Onisko, B. & Napoli, J.L. The nuclear transcription factor RARalpha associates with neuronal RNA granules and suppresses translation.
   J. Biol. Chem. 283, 20841-20847 (2008). doi:10.1074/jbc.M802314200
4. Berry, D.C., Jin, H., Majumdar, A. & Noy, N. Signaling by vitamin A and retinol-binding protein regulates gene expression to inhibit insulin responses.
   Proc. Natl Acad. Sci. USA. 108, 4340-4345 (2011). doi:10.1073/pnas.1011115108

Lipidomics: New signals to stop and protect

Lipidomics Gateway (28 September 2011) [doi:10.1038/lipidmaps.2011.27]

A lipidomics study in tandem with a functional screen has identified novel potent bioactive signals from the DHEA metabolome that inhibit leukocyte chemotaxis and confer anti-inflammatory and organ-protective properties.

Docosahexaenoic acid (DHA) has received much attention for its ability to reduce inflammation and the extent of tissue damage, particularly in the brain. These beneficial effects have so far largely been attributed to D series resolvins, such as 7,17-dihydroxydocosahexaenoic acid (7,17-diHDHA; resolvin D5), and 10,17-diHDHA, better known as neuroprotectin D1, that are biosynthesized from DHA. However, in light of recent findings by Yang et al., credit should also be given to novel bioactive products from docosahexaenoyl ethanolamide (DHEA), the ethanolamine amide of DHA.

Yang et al. began their functional metabolomics study by subjecting mouse brain extracts to liquid chromatography-tandem mass-spectrometry (LC-UV-MS-MS). In parallel, the DHEA metabolites were introduced into microfluidic chambers to assess their potential influence on the chemotaxis of human polymorphonuclear leukocytes (PMNs). The brain metabolite mixture markedly reduced interleukin-8-induced PMN chemotaxis, indicating the presence of at least one bioactive product among the metabolites.

One of the metabolites identified from the brain was 17-hydroxy-4Z,7Z,10Z,13Z,15E,19Z-docosahexaenoylethanolamide (17-HDHEA). Further analysis of the metabolic fates of 17-HDHEA using LC-UV-MS-MS-based lipidomics in mouse brain incubated with DHEA, human haemoglobin incubated with 17-hydroperoxy-DHEA (17-HpDHEA) or PMNs incubated with DHEA or 17-HpDHEA led to the identification of 4,17-diHDHEA, 7,17-diHDHEA, 10,17-diHDHEA and 15-hydroxy-16(17)-epoxydocosa-4Z,7Z,10Z,3Z,19Z-pentaenoylethanolamide (15-HEDPEA). When Yang et al. screened each of these products, 15-HEDPEA effectively induced morphology changes and inhibited PMN migration in the IL-8 gradient at 10 nM; only at higher concentrations (10 μM) did the other deoxygenated DHEA products affect chemotaxis.

To investigate how these DHEA-derived products mediated their effects, the authors tested their ability to activate cannabinoid receptors, similar to the related anandamide N-acyl-arachidonoyl-ethanolamide (AEA). Both 10,17-diHDHEA or 15-HEDPEA bound to and activated cannabinoid type 2 receptors with nanomolar affinity; they also activated type 1 receptors, although this required much higher concentrations.

Given the generation of 10,17-diHDHEA and 15-HEDPEA by PMNs, and the involvement of platelet–leukocyte interactions in haemostasis, thrombosis and inflammation, the authors investigated the functions of these two DHEA-derived products on platelet–leukocyte aggregate formation in human whole blood. Both reduced the formation of platelet–monocyte aggregates induced by platelet-activating factor (PAF) in human whole blood by ~30%, and 10,17-diHDHEA also potently inhibited PAF-stimulated platelet–PMN aggregate formation. Finally, in light of the ability of 15-HEDPEA to inhibit PMN chemotaxis and platelet–monocyte aggregate formation, its potential role in preventing second organ reperfusion injuries following ischaemia was studied. After hind limb occlusion in mice, 15-HEDPEA inhibited PMN infiltration into the lung by ~50%, thereby decreasing associated lung injury.

Yang et al. have therefore combined lipidomics with functional screening to identify potent novel bioactive molecules generated by DHEA oxidative metabolism that reduce inflammation and organ injury. This DHEA metabolome might well contribute considerably to the established anti-inflammatory and tissue-injury-reducing effects currently attributed to DHA, and highlights a potential therapeutic role for these novel molecules.

Katrin Legg

References:

ORIGINAL RESEARCH PAPER

1. Yang, R. et al. Decoding functional metabolomics with docosahexaenoyl ethanolamide (DHEA) identifies novel bioactive signals.
   J. Biol. Chem. 286, 31532-31541 (2011). doi:10.1074/jbc.M111.237990

Protein structure: Get into the groove(-tunnel)

Lipidomics Gateway (28 September 2011) [doi:10.1038/lipidmaps.2011.28]

Insights from the crystal structure of the thioesterase domain of fatty acid synthase bound by a polyunsaturated fatty acyl adduct might offer unique opportunities for the development of effective fatty acid synthase inhibitors.

Fatty acid synthase (FAS) is required for the de novosynthesis of long-chain fatty acids, predominantly palmitate, but is overexpressed, coincident with an increase in de novo fatty acid synthesis, in cancer and several other pathophysiological processes. Conversely, long-chain polyunsaturated fatty acids (PUFAs) show anti-proliferative effects, often in conjunction with inhibition of FAS activity. Now, a report by Zhang et al. of the crystal structure of the thioesterase (TE) domain of FAS with a polyunsaturated fatty acyl adduct provides insight into how PUFAs might confer their beneficial effects.

The TE domain is actually one of seven different catalytic domains in the homodimeric multifunctional FAS. Located at the carboxyl terminus, it both determines the length of the fatty acyl chain and releases the fatty acid. In their study, Zhang et al. determined the X-ray structure of the TE domain of human FAS (hFAS) covalently modified by a methylfluorophosphonate head group attached to a γ-linolenyl acyl tail (methyl γ-linolenyl fluorophosphonate (MGLFP)) at 1.48 Å resolution, revealing several binding features. Consistent with the known reactivity of MGLFP, the phosphonate head group phosphorylated a serine in the active site of TE, abrogating thioesterase activity. Unexpectedly, however, the 18-carbon polyunsaturated γ-linolenyl chain becomes buried within a long contiguous groove-tunnel site, forming complementary interactions with amino acid residues within it. This groove-tunnel is formed by a 'gatekeeper helix' or 'helix flap', an amphipathic α-helix that emerges as a consequence of ligand binding, and has not previously been observed in TE structural studies.

Because the γ-linolenyl tail bound so precisely to the TE domain, Zhang et al. surmised that other PUFAs might behave similarly, and therefore investigated the influence of three omega-6 linolenic acids — the dietary PUFAs γ-linolenic acid (GLA) and α-linolenic acid (ALA), and the GLA elongation product, dihomo-γ-linolenic acid (DGLA) — on TE domain esterase activity. DGLA inhibited TE domain activity much more effectively than GLA or ALA, although inhibition of the activity of intact hFAS by DGLA was slightly less effective compared with the isolated domain.

Next, the authors extended their studies to assess the effects of DGLA on de novo fatty acid synthesis in cells. Incubation of 3T3-L1 preadipocytes with DGLA reduced the incorporation of [¹³C]-sodium acetate into [¹³C]-palmitate by ~50%, indicating that DGLA inhibited palmitate biosynthesis. In human breast cancer cell lines, the ethyl ester of DGLA inhibited the activity of FAS, resulting in a decrease in fatty acid biosynthesis. Furthermore, the DGLA ethyl ester decreased the viability of the breast cancer cells compared with noncancerous cells, although the authors acknowledge that the precise mechanism responsible for this decrease requires further investigation.

The authors also recognize that, although their structural studies have uncovered a molecular binding mechanism for γ-linolenyl acyl chain and, by extension from the results of modelling experiments, DGLA, precisely how the TE domain binds other acyl chains, such as palmitoyl, requires a more complete atomic-level understanding. Nevertheless, the finding that polyunsaturated fatty acyl chains induce the formation of a groove-tunnel site within the active site of TE provides what the authors describe as “a tantalizing unique avenue of approach in developing FAS TE inhibitors with greater specificity and potency.”

Katrin Legg

References:

ORIGINAL RESEARCH PAPER

1. Zhang, W. et al. Crystal structure of FAS thioesterase domain with polyunsaturated fatty acyl adduct and inhibition by dihomo-γ-linolenic acid.
   Proc. Nat. Acad. Sci. USA (2011). doi:10.1073/pnas.1112334108

FURTHER READING

1. Menendez, J. A. & Lupu, R. Fatty acid synthase and the lipogenic phenotype in cancer pathogenesis.
   Nature Rev. Cancer 7, 763-777 (2007). doi:10.1038/nrc2222

Alzheimer disease: Plasma lipid biomarkers for early-stage Alzheimer disease

Nature Reviews Neurology 7, 474 (September 2011) [doi:10.1038/nrneurol.2011.125]

Changes in lipids in the brain have previously been associated with Alzheimer disease (AD). Now, an interdisciplinary team have used a multimodality lipidomics technique to measure hundreds of lipids simultaneously, and found significant changes in plasma lipids that are associated with the early stages of AD.

Animal models and postmortem analyses have identified alterations in specific brain lipids that are associated with the early stages of AD. Metabolomics—analysis of small molecules in cells and tissues—can characterize global signals of disease, but measurement in the brain is difficult, whereas peripheral plasma biomarkers are relatively easy to monitor.

Rima Kaddurah-Daouk and her colleagues aimed to analyze whether the plasma lipidome is altered in early AD, in order to “provide potential biomarkers for early diagnosis of AD, but also provide insights into the biochemical mechanisms underpinning the altered brain lipids.” AD treatments are most effective in the early stages, so biomarkers would also be useful for targeting therapeutic approaches.

In the first prospective study using multidimensional mass-spectrometry-based shotgun lipidomics, the researchers studied cellular lipids in plasma samples from 26 patients with mild or moderate AD and 26 controls. Nine lipid classes, 800 molecular species and the related pathways and networks of cellular lipids were analyzed.

The researchers focused on sphingolipids, and found that levels of eight of the 33 sphingomyelin species tested were significantly lower in patients with AD. Total sphingomyelin mass was also reduced, and ceramide content was increased. The ratios of sphingomyelin and ceramide species with identical fatty acyl chains were more-robustly able to discriminate groups than either metabolite alone.

Evidence regarding sphingolipids and AD has been inconsistent in the past, but these new results indicate that metabolomic changes vary with disease development.

The biochemical mechanisms leading to changes in peripheral blood lipids remain unknown, although it has been speculated that they are representative of changes in the brain. Kaddurah-Daouk says that she plans “to move the project forward through a study with both AD cases and apolipoprotein E genotypes using larger samples derived both centrally and peripherally. The research will move the project to both biomarker development and biochemical mechanism elucidation.”

This was a small study, and the results should be refined by further work with larger, more-diverse clinical samples. Lipidomics might be a promising new approach with which new biomarkers for AD can be identified, leading to new opportunities for treatment.

Eleanor Beal

References:

ORIGINAL RESEARCH PAPER

1. Han, X. et al. Metabolomics in early Alzheimer's disease: identification of altered plasma shingolipidome using shotgun lipidomics.
   PLoS ONE 6, e21643 (2011).

Metabolic disease: Turning 'bad' fat into 'good'

Nature Reviews Drug Discovery 10, 659 (September 2011) [doi:10.1038/nrd3540]

Excess calories are stored as fat in white adipose tissue (WAT), which — over time — leads to the development of obesity and associated metabolic diseases. Although most current obesity therapies aim to reduce caloric intake, accumulating evidence suggests that increasing cellular energy expenditure may represent a promising alternative strategy. Now, Sushil Rane and colleagues in the Diabetes, Endocrinology and Obesity Branch of the National Institute of Diabetes and Digestive and Kidney Diseases (NIDDK), US National Institutes of Health (NIH), demonstrate that blocking the transforming growth factor-β (TGFβ)–SMAD3 signalling pathway promotes the acquisition of an energy-burning brown adipose tissue (BAT) phenotype in WAT, thus ameliorating obesity and diabetes in mouse models.

TGFβ controls the development, growth and function of diverse cell types by transmitting signals via dual serine/threonine kinase receptors and transcription factors called SMADs, particularly SMAD3. Previous studies have suggested that the TGFβ–SMAD3 pathway is involved in the regulation of insulin gene transcription and pancreatic islet β-cell function. In addition, TGFβ levels have been shown to correlate positively with obesity in mice and humans. With these findings in mind, Rane and colleagues set out to determine whether TGFβ signalling has a role in the pathogenesis of metabolic disease.

First, the authors characterized Smad3-knockout mice (Smad3^−/− mice). Compared to wild-type mice, Smad3^−/−mice exhibited enhanced insulin sensitivity and were protected from diet-induced obesity, insulin resistance and hepatic steatosis. Ablation of Smad3 also reduced body weight and fat mass without reducing caloric intake, markedly impaired white adipocyte differentiation, decreased the size of adipocytes and reduced adipokine levels. In addition, it decreased the levels of inflammatory cytokines and macrophages infiltrating WAT (adipose tissue inflammation is associated with obesity). Surprisingly, WAT adopted the morphology of BAT — an energy-dissipating, thermogenic tissue that is characterized by a dense mitochondrial population — and exhibited increased expression of brown adipogenesis markers. As a result, Smad3^−/− mice were able to maintain a substantially higher body temperature even during prolonged exposure to cold, exhibited an increase in WAT mitochondrial biogenesis and function, and their metabolic rate was elevated.

Next, the authors investigated the mechanisms mediating this apparent conversion of WAT to BAT. Microarray analysis revealed that compared to control, WAT samples taken from Smad3^−/− mice displayed statistically significant increases in the expression of genes corresponding to BAT, mitochondrial function and skeletal muscle biology (BAT and skeletal muscle share developmental origins). Complementary in vitro studies indicated that this was probably mediated by the regulation of the transcriptional co-activator peroxisome proliferator-activated receptor-γ coactivator 1α and the transcription factor PRDM16, which have key roles in controlling energy metabolism and brown adipocyte development.

Finally, they examined the therapeutic relevance of their findings. In leptin-deficient ob/ob mice and mice with diet-induced obesity, administration of a TGFβ-specific neutralization antibody (1D11) reduced SMAD3 activation in WAT and conferred protection against obesity, diabetes and hepatic steatosis. Interestingly, a closely related human version of this antibody, fresolimumab, is in clinical trials of pulmonary fibrosis, renal disease and cancer.

In summary, these findings indicate a key role for the TGFβ–SMAD3 signalling pathway in adipose tissue biology. Given that TGFβ-antagonist approaches are currently in the clinic, this may be a feasible strategy to treat obesity and associated metabolic diseases.

Sarah Crunkhorn

References:

ORIGINAL RESEARCH PAPER

1. Yadav, H. et al. Protection from obesity and diabetes by blockade of TGF-β/Smad3 signalling.
   Cell Metabolism 14, 67-79 (2011).

FURTHER READING

1. Tseng, Y-H. et al. Cellular bioenergetics as a target for obesity therapy.
   Nature Rev. Drug Discov. 9, 465-482 (2010).

Researchers Identify Enzyme That Regulates Degradation of Damaged Proteins

ScienceDaily (Sep. 27, 2011) — A study by scientists at the University of California, San Diego and UC Irvine has identified an enzyme called a proteasome phosphatase that appears to regulate removal of damaged proteins from a cell. The understanding of how this process works could have important implications for numerous diseases, including cancer and Parkinson's disease. The study -- led by Jack E. Dixon, PhD, professor of Pharmacology, Cellular & Molecular Medicine, and Chemistry/Biochemistry at the University of California, San Diego and Vice President and chief scientific officer of the Howard Hughes Medical Institute -- appears in the online edition of Proceedings of the National Academy of Sciences (PNAS). Proteasomes are very large protein complexes found in all eukaryote cells, in archaea (a group of single-celled microorganisms) and in some bacteria. These basket-like chambers are essential for removing damaged or misfolded proteins from the cell. The inability of a defective proteasome to destroy misfolded or damaged proteins can be cataclysmic. Scientists have known for some time that the proteasome can be regulated by a process called phosphorylation -- a chemical process by which a phosphate is added to a protein in order to activate or deactivate it, and which plays a crucial role in biological functions, controlling nearly every cellular process, including metabolism, gene transcription and translation, cell movement, and cell death. However, researchers had a poor understanding of the kinases that put the phosphate residues on the proteasome and almost no understanding of the phosphatases that remove the phosphates. Now researchers have described for the first time how a eukaryotic phosphatase known as ubiquitin-like domain-containing C-terminal phosphatase (UBLCP1) regulates nuclear proteasome activity, revealing that UBLCP1 decreases proteasome activity by selectively dephosphorylating the proteasome. "So far, UBLCP1 is the only proteasome-specific phosphatase identified to exist in mammalian cells," said Dixon. "We are just beginning to understand how it alters proteasome activity, but one can anticipate that defects in the phosphatase activity are likely to result in major alterations in the ability of the cell to remove damaged protein." Additional contributors include first author Xing Guo, James L. Engel and Junyu Xiao, UCSD Department of Pharmacology; Vincent S. Tagliabracci, Howard Hughes Medical Institute; Xiaorong Wang and Lan Huang, UC Irvine. Funding was provided by the National Institutes of Health, a National Cancer Institute Training Grant and a Susan G. Komen postdoctoral fellowship to Guo.

College Shooting Leads to PTSD Gene Discovery

Polymorphisms in a gene that regulates serotonin may predispose individuals to the development of post-traumatic stress disorder (PTSD) if they experience or witness trauma, a longitudinal study suggested.

After a mass shooting on a college campus, young women carrying the 5-HTTLPR multimarker and rs25531 genotypes of the serotonin transporter gene exhibited significant PTSD symptom scores (P=0.03 and P=0.1, respectively), according to Kerry J. Ressler, MD, PhD, of Emory University in Atlanta, and colleagues.

In contrast, the STin2 polymorphism of the serotonin transporter gene, which has been linked with psychosis and suicidality, was not associated with PTSD symptoms (P=0.81), the researchers reported online in the Archives of General Psychiatry.

"One of the critical questions surrounding PTSD is why some individuals are at risk for developing the disorder following an index trauma while others appear to be relatively resilient," the researchers observed.

The role of genetics in PTSD was first demonstrated by twin studies among Vietnam War
veterans, but molecular genetic data have not been fully explored.

Among the earlier studies that have examined genetic links with PTSD, significant associations were most commonly found for SLC6A4, which codes for the protein responsible for regulating
serotonergic activity, 5HTT.

The so-called long/short polymorphism of this gene, 5-HTTLPR, has been shown to influence psychiatric disorders, with the shortened allele conferring heightened risks.
Another polymorphism in the same SLC6A4 gene, rs25531, is thought to modulate the expression of 5-HTTLPR, and the two polymorphisms together represent a multimarker genotype.

The researchers sought to assess the influence of this multimarker genotype, 5-HTTLPR alone, rs25531 alone, and another polymorphism in the serotonin gene referred to as STin2.

A unique set of circumstances enabled Ressler and colleagues to explore these concepts. On Feb. 14, 2008, a gunman killed five and wounded an additional 21 people on the campus of a university in northern Illinois.

Prior to this, more than 1,000 undergraduate women at the university had completed a questionnaire about sexual abuse and other traumatic experiences that could lead to PTSD, and 691 were interviewed following the shooting.

Genetic analyses for the serotonin polymorphisms were subsequently performed for 204 of the women, whose mean age was 20.

These 204 comprised the study sample.

The researchers first looked at factors that were associated with PTSD symptoms, and found significant associations with the following:

Presence in the hall where the shooting took place (P<0.05)
Hearing gunfire (P<0.005)
Seeing the gunman (P<0.001)
Seeing someone shot (P<0.05)
Being hurt in the shooting (P<0.005)
"Proximity to the shooting was highly associated with PTSD symptom severity," they observed.

Further analysis determined that participants who carried the low-expressing variant of the 5-HTTLPR multimarker had significantly higher scores on the Distressing Event Questionnaire (DEQ) after the shooting than those with the high-expressing variant (mean DEQ score of 23.91 versus mean DEQ score of 18.42, P=0.007).

When the researchers then looked at the specific symptoms of PTSD, they found greater associations with avoidance (P=0.003) than for hyperarousal (P<0.05) or nightmares and flashbacks (ns).

They noted that it has been difficult to isolate the genetic factors that contribute to PTSD because of the potential confounders of level of exposure and previous history of trauma.

Because the students in this study had already been evaluated for stress and traumatic life events, this potential confounding was minimized, they said.

Their finding implicating the 5-HTTLPR shortened alleles in PTSD risk is "biologically plausible," according to the authors, because previous research has shown that having one shortened allele reduces the expression of the serotonin protein 5HTT by 27%. The presence of a second shortened allele diminishes the expression by 30%.

Limitations of the study included the relatively small number of participants having genotype analysis, and the fact that most of the post-shooting interviews took place less than a month after the event. The DSM-IV diagnosis of PTSD requires the presence of symptoms for longer than one month.

Only 35 of the women were interviewed after one month, and associations in this subgroup were nonsignificant.

Nonetheless, the researchers argued that their data support differences in functionality of the serotonin transporter gene with PTSD risk.

"When examined in a relatively homogenous sample with shared trauma and known prior levels of child and adult trauma, the 5-HTTLPR multimarker genotype may serve as a useful predictor of risk for PTSD-related symptoms in the weeks and months following the trauma," they concluded.

By Nancy Walsh, Staff Writer, MedPage Today
Published: September 07, 2011