Untangling the relationship between diet and visceral fat mass through blood metabolomics and gut microbiome profiling.

BACKGROUND/OBJECTIVES: Higher visceral fat mass (VFM) is associated with an increased risk for developing cardio-metabolic diseases. The mechanisms by which an unhealthy diet pattern may influence visceral fat (VF) development has yet to be examined through cutting-edge multi-omic methods. Therefore, our objective was to examine the dietary influences on VFM and identify gut microbiome and metabolite profiles that link food intakes to VFM. SUBJECTS/METHODS: In 2218 twins with VFM, food intake and metabolomics data available we identified food intakes most strongly associated with VFM in 50% of the sample, then constructed and tested the 'VFM diet score' in the remainder of the sample. Using linear regression (adjusted for covariates, including body mass index and total fat mass), we investigated associations between the VFM diet score, the blood metabolomics profile and the fecal microbiome (n=889), and confirmed these associations with VFM. We replicated top findings in monozygotic (MZ) twins discordant (⩾1 s.d. apart) for VFM, matched for age, sex and the baseline genetic sequence. RESULTS: Four metabolites were associated with the VFM diet score and VFM: hippurate, alpha-hydroxyisovalerate, bilirubin (Z,Z) and butyrylcarnitine. We replicated associations between VFM and the diet score (beta (s.e.): 0.281 (0.091); P=0.002), butyrylcarnitine (0.199 (0.087); P=0.023) and hippurate (-0.297 (0.095); P=0.002) in VFM-discordant MZ twins. We identified a single species, Eubacterium dolichum to be associated with the VFM diet score (0.042 (0.011), P=8.47 × 10-5), VFM (0.057 (0.019), P=2.73 × 10-3) and hippurate (-0.075 (0.032), P=0.021). Moreover, higher blood hippurate was associated with elevated adipose tissue expression neuroglobin, with roles in cellular oxygen homeostasis (0.016 (0.004), P=9.82x10-6). CONCLUSIONS: We linked a dietary VFM score and VFM to E. dolichum and four metabolites in the blood. In particular, the relationship between hippurate, a metabolite derived from microbial metabolism of dietary polyphenols, and reduced VFM, the microbiome and increased adipose tissue expression of neuroglobin provides potential mechanistic insight into the influence of diet on VFM.

Mitogenesis and endocytosis: What's at the INTERSECTIoN?

Endocytosis is a regulated physiological process by which cell surface proteins are internalized along with extracellular factors such as nutrients, pathogens, peptides, toxins, etc. The process begins with the invagination of small regions of the plasma membrane which ultimately form intracellullar vesicles. These internalized vesicles may shuttle back to the plasma membrane to recycle the membrane components or they may be targeted for degradation. One role for endocytosis is in the attenuation of receptor signaling. For example, desensitization of activated membrane bound receptors such as G-protein coupled receptors (GPCRs) or receptor tyrosine kinases (RTKs) occurs, in part, through endocytosis of the activated receptor. However, accumulating evidence suggests that endocytosis also mediates intracellular signaling. In this review, we discuss the experimental data that implicate endocytosis as a critical component in cellular signal transduction, both in the initiation of a signal as well as in the termination of a signal. Furthermore, we focus our attention on a recently described adaptor protein, intersectin (ITSN), which provides a link to both the endocytic and the mitogenic machinery of a cell. Thus, ITSN functions at a crossroad in the biochemical regulation of cell function.

Post-transcriptional inhibition of lipopolysaccharide-induced expression of inducible nitric oxide synthase by Gö6976 in murine microglia.

Glia in the brain respond to various toxins with an increased expression of inducible nitric oxide synthase (iNOS) and an increased production of nitric oxide (NO). Here, we report that lipopolysaccharide (LPS)-induced expression of iNOS was down-regulated post-transcriptionally through the destabilization of iNOS mRNA by the indolocarbazole compound, Gö6976, in murine microglia. This Gö6976 effect is specific for iNOS since tumor necrosis factor alpha was unaffected by the compound. Interestingly, the post-transcriptional effects ascribed to Gö6976 were not observed with other inhibitors of protein kinase A, C (PKC), G, or protein tyrosine kinases. Instead, these kinases appear to affect the iNOS/NO system at the transcriptional level. In the past, Gö6976 has been reported to be a rather specific inhibitor of PKC in vitro. Results from our experiments, through prolonged treatment with phorbol esters and with the various PKC inhibitors including phorbol ester-insensitive PKC isotype inhibitor, suggest that the Gö6976-mediated post-transcriptional regulation of iNOS gene expression and NO production in microglia is not mediated through its reputed effects on PKC activity. Since the effects of various neurotoxins and certain neurodegenerative diseases may be manifested through alterations in the iNOS/NO system, post-transcriptional control of this system may represent a novel strategy for therapeutic intervention.

Galanin expression is decreased by cAMP-elevating agents in cultured sympathetic ganglia.

Galanin expression is co-regulated in peripheral neurons with that of vasoactive intestinal peptide (VIP) under a variety of conditions. For example, the expression of both increase after explantation of adult rat superior cervical ganglia (SCG). Because VIP participates in a positive feedback loop regulating its own expression, we examined whether VIP also increases galanin expression. Galanin mRNA and peptide are nearly undetectable in the SCG in vivo, but increase dramatically after 24-48 h in organ culture. Addition of VIP or forskolin to the culture medium reduced galanin mRNA expression by 75% and 77%, respectively, and reduced galanin peptide expression by 76% and 82%, respectively, compared with ganglia cultured in control medium. In contrast, isoproterenol stimulation did not significantly alter levels of galanin mRNA or peptide, consistent with previous observations that isoproterenol exerts its effect on SCG non-neuronal cells, but not on neurons. The results indicate that galanin and VIP are differentially regulated in sympathetic neurons by cAMP- elevating agents.

Vasoactive intestinal peptide enhances its own expression in sympathetic neurons after injury.

Neurons in the adult rat superior cervical sympathetic ganglion (SCG) dramatically increase their content of vasoactive intestinal peptide (VIP) and its mRNA after axotomy in vivo and after explantation. Because the VIP gene contains a functional cAMP response element, the effects of cAMP-elevating agents on VIP expression were examined. VIP, forskolin, or isoproterenol increased cAMP accumulation in explanted ganglia. Secretin, a peptide chemically related to VIP, or forskolin increased VIP levels above those seen in ganglia cultured in control medium, whereas treatment with VIP or secretin increased the level of peptide histidine isoleucine (PHI), a peptide coded for by the same mRNA that encodes VIP. VIP or forskolin also increased VIP-PHI mRNA. In contrast, isoproterenol did not alter levels of VIP, PHI, or VIP-PHI mRNA. Although VIP or forskolin increased cAMP levels in both dissociated neurons and in non-neuronal cells, isoproterenol significantly stimulated cAMP accumulation only in the latter. VIP6-28 was an effective antagonist of the actions of exogenous VIP on cAMP and VIP-PHI mRNA in neuron-enriched cultures. When adult SCG explants were cultured in defined medium, endogenous VIP immunoreactivity was released. When VIP6-28 was added to such cultures, it significantly inhibited the increase in VIP-PHI mRNA that normally occurs. These data indicate that VIP, or a closely related molecule, produced by adult neurons after injury can enhance the expression of VIP. Such a mechanism may prolong the period during which VIP is elevated after axonal damage. The possibility is also discussed that, because VIP is present in preganglionic neurons in normal animals, its release during periods of increased sympathetic nerve activity could alter VIP expression in the SCG.

Galanin and vasoactive intestinal peptide messenger RNAs increase following axotomy of adult sympathetic neurons.

The adult rat superior cervical ganglion (SCG) contains low levels of galanin- and vasoactive intestinal peptide-(VIP) like immunoreactivity, with very few immunostained principal neurons. Immunoreactivity for both neuropeptides increases in these neurons after explantation or postganglionic axotomy in vivo. Northern blot analysis has demonstrated concomitant increases in mRNAs encoding these peptides. To localize cells in axotomized ganglia which increase their expression of these mRNAs, we performed in situ hybridization studies. In control SCG, only a few principal neurons contained mRNA for either galanin or VIP. After 48 h in organ culture, galanin mRNA was expressed in the majority of principal neurons. At 48 h after in vivo axotomy of both postganglionic trunks of the SCG, the internal and external carotid nerves, the distribution and number of neurons, expressing galanin mRNA increased similarly to that seen in culture. Lesioning either trunk alone produced increases in galanin mRNA localized to those regions of the ganglion containing neurons that project into the lesioned trunk. Transection of the predominantly preganglionic cervical sympathetic trunk increased galanin mRNA expression in a small population of neurons near that nerve trunk. The distributions of these labeled neurons, together with previous neuroanatomical studies, suggests that they had been axotomized by the lesions. Similar studies examining VIP mRNA expression demonstrated that although considerably fewer VIP mRNA expressing neurons than galanin mRNA expressing neurons were present after axotomy, the distribution of neuropeptide mRNA-positive cells were similar in both cases. These observations suggest that increases in the peptides galanin and VIP after nerve transection result from changes in the levels of their mRNAs in those neurons that have been axotomized.

Regional difference in susceptibility to lipopolysaccharide-induced neurotoxicity in the rat brain: role of microglia.

Inflammation in the brain has been increasingly associated with the development of a number of neurological diseases. The hallmark of neuroinflammation is the activation of microglia, the resident brain immune cells. Injection of bacterial endotoxin lipopolysaccharide (LPS) into the hippocampus, cortex, or substantia nigra of adult rats produced neurodegeneration only in the substantia nigra. Although LPS appeared to impact upon mesencephalic neurons in general, an extensive loss of dopaminergic neurons was observed. Analysis of the abundance of microglia revealed that the substantia nigra had the highest density of microglia. When mixed neuron-glia cultures derived from the rat hippocampus, cortex, or mesencephalon were treated with LPS, mesencephalic cultures became sensitive to LPS at a concentration as low as 10 ng/ml and responded in a dose-dependent manner with the production of inflammatory factors and a loss of dopaminergic and other neurons. In contrast, hippocampal or cortical cultures remained insensitive to LPS treatment at concentrations as high as 10 microg/ml. Consistent with in vivo observations, mesencephalic cultures had fourfold to eightfold more microglia than cultures from other regions. The positive correlation between abundance of microglia and sensitivity to LPS-induced neurotoxicity was further supported by the observation that supplementation with enriched microglia derived from mesencephalon or cortex rendered LPS-insensitive cortical neuron-glia cultures sensitive to LPS-induced neurotoxicity. These data indicate that the region-specific differential susceptibility of neurons to LPS is attributable to differences in the number of microglia present within the system and may reflect levels of inflammation-related factors produced by these cells.

Glycoinositol phospholipid anchor-defective K562 mutants with biochemical lesions distinct from those in Thy-1- murine lymphoma mutants.

Deficient expression of glycoinositol phospholipid (GPI)-anchored surface proteins has been linked to six different genetic defects in Thy-1- murine lymphoma mutants. In this study, human K562 cell mutants defective in GPI anchoring were derived by anti-decay-accelerating factor (CD55) based negative fluorescent cell sorting of N-methyl-N'-nitro-N-nitrosoguanidine pretreated cells. Homologous cell fusions of six clones that complemented a previously described K562 mutant corresponding to one of the Thy-1- mutant classes (Hirose, S., Mohney, R. P., Mutka, S. C., Ravi, L., Singleton, D. R., Perry, G., Tartakoff, A., and Medof, M. E. (1992) J. Biol. Chem. 267, 5272-5278) showed that they segregated into two complementation groups. In heterologous cell fusions, representative clones of each group complemented Thy-1 expression by all of the previously described GPI anchor pathway-defective Thy-1- murine lymphoma classes (A, B, C, E, F, and H) but not class(es) D (and I) defective in the Thy-1 structural gene. Analyses of putative GPI anchor precursors synthesized by the two lines revealed that one mutant exhibited a complete block in deacetylation of N-acetyl-D-glucosamine-inositol phospholipid to glucosamine (GlcN)-inositol phospholipid, whereas the other mutant assembled GlcN-inositol phospholipid and subsequent mannose (Man)-containing intermediates but showed markedly increased amounts of the terminal ethanolamine (EthN)-phosphate (P)-substituted putative anchor precursors, EthN-P-6ManMan(EthN-P-->)ManGlcN- and EthN-P-6Man(EthN-P-6)Man(EthN- P-->)ManGlcN-acylinositol phospholipid (H7 and H8). We designate these new complementation classes J, harboring a defect in N-acetyl-D-glucosamine-inositol phospholipid deacetylation, and K, deficient in a step preliminary to or associated with protein transfer of assembled anchor precursors. The availability of these new mutant classes should aid in characterization of the GPI anchor pathway enzymes providing for these reactions.

Derivation and characterization of glycoinositol-phospholipid anchor-defective human K562 cell clones.

To aid in studies of human glycoinositol-phospholipid (GPI) anchor pathway biochemistry in normal and affected paroxysmal nocturnal hemoglobinuria cells, GPI anchor-defective human K562 cell lines were derived by negative fluorescent sorting of anti-decay-accelerating factor (DAF) monoclonal antibody-stained cells either following or in the absence of ethylmethylsulfonate pretreatment. The resulting cloned cells showed deficiencies of both DAF and GPI-anchored CD59, some (designated group A) exhibiting total absence and some (designated group B) exhibiting approximately 10% levels of surface expression of the two proteins. In heterologous cell fusions, group A clones complemented defective Thy-1 expression by class A, B, C, E, and I Thy-1-negative lymphoma lines, but not H or D lines, the latter of which is defective in the Thy-1 structural gene. In contrast, group B clones complemented all previously described GPI anchor pathway-defective lymphoma classes. Immunoradiomatic assays of cells and supernatants and 35S biosynthetic labeling showed that group A cells degraded DAF protein while group B cells secreted it but failed to attach a GPI anchor structure. [3H]Man labeling of intact cells and UDP-[3H]GlcNAc and GDP-[3H]Man labeling of broken cell preparations demonstrated that group A cells failed to synthesize GlcNAc- and GlcN-PI (GPI-A and -B) as well as more polar mannolipids, whereas group B cells showed accumulation of GlcNAc-PI with approximately 10-fold diminished levels of GlcN-PI and more polar mannolipids. The failed assembly of GlcNAc-PI in group A cells and the reduced conversion of this intermediate to GlcN-PI in group B cells indicates that the former harbors a defect in UDP-GlcNAc transferase or in assembly of its PI acceptor, while the latter harbors a defect in GlcN-PI deacetylase activity.

Changes in neuropeptide phenotype after axotomy of adult peripheral neurons and the role of leukemia inhibitory factor.

Adult peripheral neurons undergo dramatic shifts in gene expression following axotomy that are collectively referred to as the cell body reaction. Changes in neuropeptide expression are a prominent feature of these axotomized neurons. For example, while sympathetic, sensory, and motor neurons do not normally express the neuropeptides galanin and vasoactive intestinal peptide, they begin to do so within days after axotomy. In contrast, the expression of other peptides, which these neurons normally express, such as neuropeptide Y in sympathetic neurons and substance P in sensory neurons, is decreased. Recent studies in sympathetic neurons have demonstrated that leukemia inhibitory factor plays an important role in triggering these changes in neuropeptide phenotype in adult neurons. Future studies will be directed at determining to what extent LIF triggers the many other changes in gene expression after sympathetic axotomy and whether this cytokine plays a similar role in sensory and motor neurons.