Gut microbiome and serum metabolome analyses identify molecular biomarkers and altered glutamate metabolism in fibromyalgia.

BACKGROUND: Fibromyalgia is a complex, relatively unknown disease characterised by chronic, widespread musculoskeletal pain. The gut-brain axis connects the gut microbiome with the brain through the enteric nervous system (ENS); its disruption has been associated with psychiatric and gastrointestinal disorders. To gain an insight into the pathogenesis of fibromyalgia and identify diagnostic biomarkers, we combined different omics techniques to analyse microbiome and serum composition. METHODS: We collected faeces and blood samples to study the microbiome, the serum metabolome and circulating cytokines and miRNAs from a cohort of 105 fibromyalgia patients and 54 age- and environment-matched healthy individuals. We sequenced the V3 and V4 regions of the 16S rDNA gene from faeces samples. UPLC-MS metabolomics and custom multiplex cytokine and miRNA analysis (FirePlex™ technology) were used to examine sera samples. Finally, we combined the different data types to search for potential biomarkers. RESULTS: We found that the diversity of bacteria is reduced in fibromyalgia patients. The abundance of the Bifidobacterium and Eubacterium genera (bacteria participating in the metabolism of neurotransmitters in the host) in these patients was significantly reduced. The serum metabolome analysis revealed altered levels of glutamate and serine, suggesting changes in neurotransmitter metabolism. The combined serum metabolomics and gut microbiome datasets showed a certain degree of correlation, reflecting the effect of the microbiome on metabolic activity. We also examined the microbiome and serum metabolites, cytokines and miRNAs as potential sources of molecular biomarkers of fibromyalgia. CONCLUSIONS: Our results show that the microbiome analysis provides more significant biomarkers than the other techniques employed in the work. Gut microbiome analysis combined with serum metabolomics can shed new light onto the pathogenesis of fibromyalgia. We provide a list of bacteria whose abundance changes in this disease and propose several molecules as potential biomarkers that can be used to evaluate the current diagnostic criteria.

Sex differences in LXR expression in normal offspring and in rats born to diabetic dams.

Gestational diabetes (GD) alters normal fetal development and is related to a diabetogenic effect in the progeny. Liver X receptors (LXRs) are considered to be potential drug targets for the regulation, treatment, or prevention of diabetes. The aim of this study was to evaluate early and late changes of LXR in the hippocampus and hypothalamus of the male and female offspring of control (CO) and diabetic (DO) mothers. We used an experimental model of streptozotocin-induced GD to assess the protein expression of LXRα (NR1H3) and LXRß (NR1H2) by western blotting. The tissues were obtained from CO and DO animals at postnatal day 1 (1D), day 10 (10D), and day 35 (35D) and 9 months (9M). In CO, the LXR expression showed significant differences among the groups, which were tissue- and receptor-specific (P<0.05). Sex differences in CO were found only in the hypothalamus for LXRß expression at 35D and 9M (P<0.05). When CO and DO were compared, differences between them were observed in the majority of the studied groups at 1D (male hippocampus, LXRα 31% and LXRß 161%; female hippocampus, LXRß 165%; male hypothalamus, LXRß 182%; and female hypothalamus, LXRα 85%; P<0.05). However, these differences disappeared later with the exception of LXRß expression in the male hypothalamus (P<0.05). The area under the curve during the glucose tolerance test correlated negatively with LXRß in CO but not in DO animals. Moreover, in a male DO subpopulation this correlation was positive as it occurs in intolerant animals. These results indicate that GD affects hypothalamic LXR expression differently in male and female offspring.

Alterations of LXRα and LXRß expression in the hypothalamus of glucose-intolerant rats.

Liver X receptor (LXR) α and ß are nuclear receptors that are crucial for the regulation of carbohydrate and lipid metabolism. Activation of LXRs in the brain facilitates cholesterol clearance and improves cognitive deficits, thus they are considered as promising drug targets to treat diseases such as atherosclerosis and Alzheimer's disease. Nevertheless, little is known about the function and localization of LXRs in the brain. Here, we studied the expression of LXR in the brains of rats that received free access to 10% (w/v) fructose group (FG) in their beverages or water control drinks (control group (CG)). After 6 weeks rats in the FG presented with hypertriglyceridemia, hyperinsulinemia, and became glucose intolerant, suggesting a progression toward type 2 diabetes. We found that hypothalamic LXR expression was altered in fructose-fed rats. Rats in the FG presented with a decrease in LXRß levels while showing an increase in LXRα expression in the hypothalamus but not in the hippocampus, cerebellum, or neocortex. Moreover, both LXRα and ß expression correlated negatively with insulin and triglyceride levels. Interestingly, LXRß showed a negative correlation with the area under the curve during the glucose tolerance test in the CG and a positive correlation in the FG. Immunocytochemistry revealed that the paraventricular and ventromedial nuclei express mainly LXRα whereas the arcuate nucleus expresses LXRß. Both LXR immunosignals were found in the median preoptic area. This is the first study showing a relationship between glucose and lipid homeostasis and the expression of LXRs in the hypothalamus, suggesting that LXRs may trigger neurochemical and neurophysiological responses for the control of food intake and energy expenditure through these receptors.

Down regulation of the proliferation and apoptotic pathways in the embryonic brain of diabetic rats.

Compelling evidence shows that the offspring subjected to uncontrolled hyperlycemia during gestation display behavioral, neurochemical, and cellular abnormalities during adulthood. However, the molecular mechanisms underlying these defects remain elusive. Previous studies have shown an increased rate of apoptosis and a decreased index of neuronal proliferation associated with diabetic embryopathy. The aim of the present study was to determine whether impairments in apoptotic related proteins also occur in the developing central nervous system from non-malformed embryos exposed to uncontrolled gestational hyperglycemia. Pregnant rats injected with either streptozotocin or vehicle were killed on gestational day 19. Offspring brains were quickly removed to evaluate protein expression by Western blotting. Embryonic brains from diabetic rats exhibited a decrease in the cell survival p-Akt expression (52.83 ± 24.35%) and in the pro-apoptotic protein Bax (56.16 ± 6.47%). Moreover, the anti-apoptotic protein Bcl-2 showed a non-significant increase while there were no changes in Procaspase 3 or cleaved Caspase 3 proteins. The cytoskeleton proteins NF-200 and GFAP were also examined. Neither NF-200 nor GFAP showed differences in embryonic brains from diabetic rats compared to controls. Altogether, these results indicate that both proliferation and apoptotic pathways are decreased in the brain from the developing offspring of diabetic rats. Since selective neuronal apoptosis, as well as selective cell proliferation, are specifically involved in brain organogenesis, it is possible that simultaneous impairments during the perinatal period contribute to the long lasting alterations observed in the adult brain.

Age-related changes of the GABA-B receptor in the lumbar spinal cord of male rats and penile erection.

Dorsal horn neurons of lumbosacral spinal cord innervate penile vasculature and regulate penile erection. GABAergic system is involved in the regulation of male sexual behavior. Because aging is frequently accompanied by a progressive decline in erectile function, the aim of this work was to examine age-related changes of the GABA-B receptor in the lumbar spinal cord. Sprague-Dawley rats of 10 and 21 days old, 3, 9 and 20 months old were used. GABA-B receptors were evaluated by quantitative autoradiography using [3H]-Baclofen as ligand with or without GABA (10 microM) to determine the non-specific binding. Ten days after birth a homogeneous neuroanatomical distribution pattern was found in the gray matter, however at 20-day-old adult distribution emerged becoming heterogeneous with the highest binding values at layers II-III and X. In dorsal layers a significant decrease was observed in 9-month-old rats while layer X showed an earlier decrease (21-day-old). GABA-B receptor affinity showed significant age-dependent and regional increase. The GABA-B receptor decrease in aged rats seems not to be related to this receptor inhibitory function in penile erection. Moreover the changes found in GABA-B receptor binding anatomical distribution may indicate its role in the morphological development of the lumbar spinal cord rather than in the decline of the erectile function.

A thermodynamic and kinetic analysis of the folding pathway of an SH3 domain entropically stabilised by a redesigned hydrophobic core.

The folding thermodynamics and kinetics of the alpha-spectrin SH3 domain with a redesigned hydrophobic core have been studied. The introduction of five replacements, A11V, V23L, M25V, V44I and V58L, resulted in an increase of 16% in the overall volume of the side-chains forming the hydrophobic core but caused no remarkable changes to the positions of the backbone atoms. Judging by the scanning calorimetry data, the increased stability of the folded structure of the new SH3-variant is caused by entropic factors, since the changes in heat capacity and enthalpy upon the unfolding of the wild-type and mutant proteins were identical at 298 K. It appears that the design process resulted in an increase in burying both the hydrophobic and hydrophilic surfaces, which resulted in a compensatory effect upon the changes in heat capacity and enthalpy. Kinetic analysis shows that both the folding and unfolding rate constants are higher for the new variant, suggesting that its transition state becomes more stable compared to the folded and unfolded states. The phi(double dagger-U) values found for a number of side-chains are slightly lower than those of the wild-type protein, indicating that although the transition state ensemble (TSE) did not change overall, it has moved towards a more denatured conformation, in accordance with Hammond's postulate. Thus, the acceleration of the folding-unfolding reactions is caused mainly by an improvement in the specific and/or non-specific hydrophobic interactions within the TSE rather than by changes in the contact order. Experimental evidence showing that the TSE changes globally according to its hydrophobic content suggests that hydrophobicity may modulate the kinetic behaviour and also the folding pathway of a protein.

Conformational strain in the hydrophobic core and its implications for protein folding and design.

We have designed de novo 13 divergent spectrin SH3 core sequences to determine their folding properties. Kinetic analysis of the variants with stability similar to that of the wild type protein shows accelerated unfolding and refolding rates compatible with a preferential stabilization of the transition state. This is most likely caused by conformational strain in the native state, as deletion of a methyl group (Ile-->Val) leads to deceleration in unfolding and increased stability (up to 2 kcal x mol(-1)). Several of these Ile-->Val mutants have negative phi(-U) values, indicating that some noncanonical phi(-U) values might result from conformational strain. Thus, producing a stable protein does not necessarily mean that the design process has been entirely successful. Strained interactions could have been introduced, and a reduction in the buried volume could result in a large increase in stability and a reduction in unfolding rates.