Impact of Bariatric Surgery on Circulating Metabolites and Cognitive Performance.

INTRODUCTION: Bariatric surgery is an effective intervention to reduce obesity and improve associated comorbidities. However, its effects on cognitive function are still the subject of debate. Given that the bioavailability of circulating metabolites can influence brain metabolism and cognitive performance, we aimed to assess the effects of bariatric surgery on plasma metabolic profiles and cognitive performance. METHODS: We recruited 26 women undergoing gastric bypass surgery. We conducted anthropometric assessments and collected plasma samples for metabolomic analysis. A set of 4 cognitive tests were used to evaluate cognitive performance. Participants were reevaluated 1 year post-surgery. RESULTS: After surgery, attention capacity and executive function were improved, while immediate memory had deteriorated. Regarding metabolic profile, reduction of beta-tocopherol and increase of serine, glutamic acid, butanoic acid, and glycolic acid were observed. To better understand the relationship between cognitive function and metabolites, a cluster analysis was conducted to identify more homogeneous subgroups based on the cognitive performance. We identified cluster 1, which did not show changes in cognitive performance after surgery, and cluster 2, which showed improved attention and executive function, but reduced performance in the immediate memory test. Thus, cluster 2 was more homogeneous group that replicated the results of non-clustered subjects. Analysis of the metabolic profile of cluster 2 confirmed serine, glutamic acid, and glycolic acid as potential metabolites associated with cognitive performance. CONCLUSIONS: Metabolites identified in this study have potential for biomarkers and alternative therapeutic target to prevent obesity-related cognitive decline. KEY POINTS: • Attention capacity and executive function were improved 12 months post bariatric surgery. • Immediate memory was worsened 12 months post bariatric surgery. • Serine, glutamic acid, and glycolic acid are potential metabolites linked to the alteration of cognitive performance.

Severe Obesity in Women Can Lead to Worse Memory Function and Iron Dyshomeostasis Compared to Lower Grade Obesity.

Objective: Obesity is one of the modifiable risk factors for dementia. Insulin resistance, the abundance of advanced glycated end-products, and inflammation are some of the mechanisms associated with the lower cognitive performance observed in obesity. This study aims to evaluate the cognitive function of subjects with distinct degrees of obesity, comparing class I and II obesity (OBI/II) to class III obesity (OBIII), and to investigate metabolic markers that can distinguish OBIII from OBI/II. Study Design. This is a cross-sectional study, in which 45 females with BMI varying from 32.8 to 51.9 kg/m2 completed a set of 4 cognitive tests (verbal paired-associate test, stroop color, digit span, and Toulouse-Pieron cancellation test) and their plasma metabolites, enzymes, and hormones related to glycemia, dyslipidemia, and liver function, as well as the biomarkers of iron status, were concomitantly analyzed. Results: OBIII showed lower scores in the verbal paired-associate test compared to OBI/II. In other cognitive tests, both groups showed similar performance. OBIII presented a lower iron status compared to OBI/II based on total iron binding capacity, degree of transferrin saturation, hemoglobin, mean corpuscular volume, and mean corpuscular hemoglobin. The levels of indicators for glycemia, liver function, and lipid metabolism were similar in both groups. Analysis of plasma metabolites showed that OBIII had lower levels of pyroglutamic acid, myoinositol, and aspartic acid and higher levels of D-ribose than OBI/II. Conclusion: Iron is an essential micronutrient for several metabolic pathways. Thus, iron dyshomeostasis observed in severe obesity may aggravate the cognitive impairment by altering metabolic homeostasis and enhancing oxidative stress. These findings can contribute to searching for biomarkers that indicate cognitive performance in the population with obesity.

Iron-deficient diet induces distinct protein profile related to energy metabolism in the striatum and hippocampus of adult rats.

Iron deficiency is a public health problem that affects all age groups. Its main consequence is anemia, but it can also affect cognitive functions. Although the negative effects of iron deficiency on cognitive function have been extensively described, the underlying mechanism has not been fully investigated. Thus, to gain an unbiased insight into the effects of iron deficiency (ID) on discrete brain regions, we performed a proteomic analysis of the striatum and hippocampus of adult rats subjected to an iron restricted (IR) diets for 30 days. We found that an IR diet caused major alterations in proteins related to glycolysis and lipid catabolism in the striatum. In the hippocampus, a larger portion of proteins related to oxidative phosphorylation and neurodegenerative diseases were altered. These alterations in the striatum and hippocampus occurred without a reduction in local iron levels, although there was a drastic reduction in liver iron and ferritin. Moreover, the IR group showed higher fasting glycaemia than the control group. These results suggest that brain iron content is preserved during acute iron deficiency, but the alterations of other systemic metabolites such as glucose may trigger distinct metabolic adaptations in each brain region. Abnormal energy metabolism precedes and persists in many neurological disorders. Thus, altered energy metabolism can be one of the mechanisms by which iron deficiency affects cognitive functions.

Sleep deprivation regulates availability of PrPC and Aß peptides which can impair interaction between PrPC and laminin and neuronal plasticity.

PrPC is a glycoprotein capable to interact with several molecules and mediates diverse signaling pathways. Among numerous ligands, laminin (LN) is known to promote neurite outgrowth and memory consolidation, while amyloid-beta oligomers (Aßo) trigger synaptic dysfunction. In both pathways, mGluR1 is recruited as co-receptor. The involvement of PrPC /mGluR1 in these opposite functions suggests that this complex is a key element in the regulation of synaptic activity. Considering that sleep-wake cycle is important for synaptic homeostasis, we aimed to investigate how sleep deprivation affects the expression of PrPC and its ligands, laminin, Aßo, and mGluR1, a multicomplex that can interfere with neuronal plasticity. To address this question, hippocampi of control (CT) and sleep deprived (SD) C57BL/6 mice were collected at two time points of circadian period (13 hr and 21 hr). We observed that sleep deprivation reduced PrPC and mGluR1 levels with higher effect in active state (21 hr). Sleep deprivation also caused accumulation of Aß peptides in rest period (13 hr), while laminin levels were not affected. In vitro binding assay showed that Aßo can compete with LN for PrPC binding. The influence of Aßo was also observed in neuritogenesis. LN alone promoted longer neurite outgrowth than non-treated cells in both Prnp+/+ and Prnp0/0 genotypes. Aßo alone did not show any effects, but when added together with LN, it attenuated the effects of LN only in Prnp+/+ cells. Altogether, our findings indicate that sleep deprivation regulates the availability of PrPC and Aß peptides, and based on our in vitro assays, these alterations induced by sleep deprivation can negatively affect LN-PrPC interaction, which is known to play roles in neuronal plasticity.

Iron-Restricted Diet Affects Brain Ferritin Levels, Dopamine Metabolism and Cellular Prion Protein in a Region-Specific Manner.

Iron is an essential micronutrient for several physiological functions, including the regulation of dopaminergic neurotransmission. On the other hand, both iron, and dopamine can affect the folding and aggregation of proteins related with neurodegenerative diseases, such as cellular prion protein (PrPC) and α-synuclein, suggesting that deregulation of iron homeostasis and the consequential disturbance of dopamine metabolism can be a risk factor for conformational diseases. These proteins, in turn, are known to participate in the regulation of iron and dopamine metabolism. In this study, we evaluated the effects of dietary iron restriction on brain ferritin levels, dopamine metabolism, and the expression levels of PrPC and α-synuclein. To achieve this goal, C57BL/6 mice were fed with iron restricted diet (IR) or with normal diet (CTL) for 1 month. IR reduced iron and ferritin levels in liver. Ferritin reduction was also observed in the hippocampus. However, in the striatum of IR group, ferritin level was increased, suggesting that under iron-deficient condition, each brain area might acquire distinct capacity to store iron. Increased lipid peroxidation was observed only in hippocampus of IR group, where ferritin level was reduced. IR also generated discrete results regarding dopamine metabolism of distinct brain regions: in striatum, the level of dopamine metabolites (DOPAC and HVA) was reduced; in prefrontal cortex, only HVA was increased along with the enhanced MAO-A activity; in hippocampus, no alterations were observed. PrPC levels were increased only in the striatum of IR group, where ferritin level was also increased. PrPC is known to play roles in iron uptake. Thus, the increase of PrPC in striatum of IR group might be related to the increased ferritin level. α-synuclein was not altered in any regions. Abnormal accumulation of ferritin, increased MAO-A activity or lipid peroxidation are molecular features observed in several neurological disorders. Our findings show that nutritional iron deficiency produces these molecular alterations in a region-specific manner and provide new insight into the variety of molecular pathways that can lead to distinct neurological symptoms upon iron deficiency. Thus, adequate iron supplementation is essential for brain health and prevention of neurological diseases.

Dopamine induces the accumulation of insoluble prion protein and affects autophagic flux.

Accumulation of protein aggregates is a histopathological hallmark of several neurodegenerative diseases, but in most cases the aggregation occurs without defined mutations or clinical histories, suggesting that certain endogenous metabolites can promote aggregation of specific proteins. One example that supports this hypothesis is dopamine and its metabolites. Dopamine metabolism generates several oxidative metabolites that induce aggregation of α-synuclein, and represents the main etiology of Parkinson's diseases. Because dopamine and its metabolites are unstable and can be highly reactive, we investigated whether these molecules can also affect other proteins that are prone to aggregate, such as cellular prion protein (PrP(C)). In this study, we showed that dopamine treatment of neuronal cells reduced the number of viable cells and increased the production of reactive oxygen species (ROS) as demonstrated in previous studies. Overall PrP(C) expression level was not altered by dopamine treatment, but its unglycosylated form was consistently reduced at 100 µM of dopamine. At the same concentration, the level of phosphorylated mTOR and 4EBP1 was also reduced. Moreover, dopamine treatment decreased the solubility of PrP(C), and increased its accumulation in autophagosomal compartments with concomitant induction of LC3-II and p62/SQSTM1 levels. In vitro oxidation of dopamine promoted formation of high-order oligomers of recombinant prion protein. These results suggest that dopamine metabolites alter the conformation of PrP(C), which in turn is sorted to degradation pathway, causing autophagosome overload and attenuation of protein synthesis. Accumulation of PrP(C) aggregates is an important feature of prion diseases. Thus, this study brings new insight into the dopamine metabolism as a source of endogenous metabolites capable of altering PrP(C) solubility and its subcellular localization.

Metabotropic glutamate receptors transduce signals for neurite outgrowth after binding of the prion protein to laminin γ1 chain.

The prion protein (PrP(C)) is highly expressed in the nervous system, and its abnormal conformer is associated with prion diseases. PrP(C) is anchored to cell membranes by glycosylphosphatidylinositol, and transmembrane proteins are likely required for PrP(C)-mediated intracellular signaling. Binding of laminin (Ln) to PrP(C) modulates neuronal plasticity and memory. We addressed signaling pathways triggered by PrP(C)-Ln interaction in order to identify transmembrane proteins involved in the transduction of PrP(C)-Ln signals. The Ln γ1-chain peptide, which contains the Ln binding site for PrP(C), induced neuritogenesis through activation of phospholipase C (PLC), Ca(2+) mobilization from intracellular stores, and protein kinase C and extracellular signal-regulated kinase (ERK1/2) activation in primary cultures of neurons from wild-type, but not PrP(C)-null mice. Phage display, coimmunoprecipitation, and colocalization experiments showed that group I metabotropic glutamate receptors (mGluR1/5) associate with PrP(C). Expression of either mGluR1 or mGluR5 in HEK293 cells reconstituted the signaling pathways mediated by PrP(C)-Ln γ1 peptide interaction. Specific inhibitors of these receptors impaired PrP(C)-Ln γ1 peptide-induced signaling and neuritogenesis. These data show that group I mGluRs are involved in the transduction of cellular signals triggered by PrP(C)-Ln, and they support the notion that PrP(C) participates in the assembly of multiprotein complexes with physiological functions on neurons.

The influence of obstructive sleep apnea on the expression of glycerol-3-phosphate dehydrogenase 1 gene.

Glycerol-3-phosphate dehydrogenase 1 (GPD1) is considered to be a key enzyme that connects carbohydrate and lipid metabolism. This gene is induced in response to sleep deprivation, suggesting a potential role for this enzyme in the manifestation of obstructive sleep apnea (OSA). This study aims to examine the effects of sleep apnea, obesity and other relevant clinical parameters on GPD1 expression in the peripheral blood of a rigorously selected sample population in order to identify a biological marker that would allow for early intervention and prevention of the disorder. Clinical and sleep parameters were assessed by a complete full-night polysomnography and the expression of GPD1 at the mRNA level was determined. The results were compared among 20 OSA patients and 20 controls, further classified into two subgroups according to their body mass index. The expression levels of the GPD1 gene did not differ between patients with OSA and their matched controls. The results were not affected by the clinical and biochemical measurements, the sleep parameters or the severity of nocturnal hypoxemia. On the other hand, individuals with OSA had higher levels of fasting glucose when compared with weight-matched controls (P = 0.01). Moreover, higher very low-density lipoprotein (VLDL) was found in the over-weight OSA patient group, and higher cholesterol levels were found in the eutrophic OSA group when compared with their respective controls (P < 0.05). Based on logistic regression analyses, fasting glucose levels emerged as an independent factor for OSA in both the eutrophic (odds ratio [OR] = 1.27; 95% confidence interval [CI] = 1.00-1.59) and over-weight groups (OR = 1.29; 95% CI = 1.04-1.59). Although the results from the current study corroborate the growing body of data connecting OSA to altered glucose metabolism, it does not provide evidence for the modulation of GPD1 transcription by either OSA or its related phenotypes. This suggests that GPD1 may not play a major role in the OSA manifestation.

Altered sleep patterns and physiologic characteristics in spontaneous dwarf rats.

Spontaneous dwarf rats are a useful experimental model for studying various biologic events associated with pituitary dwarfism. Dwarf rats occurred serendipitously in our colony of Wistar rats during experimental breeding. This study aimed to describe the sleep pattern and physiologic characteristics of these rats compared with normal-sized adult rats. Because growth hormone can attenuate the upregulation of ceruloplasmin expression caused by acute inflammation, we also assessed the basal levels of serum ceruloplasmin in these animals. At 90 d of age, body weight and length were significantly lower in dwarf rats relative to normal rats. Dwarves had lower concentrations of serum testosterone and growth hormone, but progesterone was unchanged. Corticosterone levels did not differ between groups. During the light period, the percentage of sleep time recorded and duration of slow-wave sleep did not differ between groups. However, compared with controls, dwarf rats had marked fragmentation of sleep and less paradoxical sleep. During the dark phase, sleep patterns in dwarf rats were within the normal range. Immunoblotting data showed that the levels of ceruloplasmin in serum were lower in dwarf rats. Our findings provide insight into pathologic processes related to growth hormone deficiency.

Validation of commonly used reference genes for sleep-related gene expression studies.

BACKGROUND: Sleep is a restorative process and is essential for maintenance of mental and physical health. In an attempt to understand the complexity of sleep, multidisciplinary strategies, including genetic approaches, have been applied to sleep research. Although quantitative real time PCR has been used in previous sleep-related gene expression studies, proper validation of reference genes is currently lacking. Thus, we examined the effect of total or paradoxical sleep deprivation (TSD or PSD) on the expression stability of the following frequently used reference genes in brain and blood: beta-actin (b-actin), beta-2-microglobulin (B2M), glyceraldehyde-3-phosphate dehydrogenase (GAPDH), and hypoxanthine guanine phosphoribosyl transferase (HPRT). RESULTS: Neither TSD nor PSD affected the expression stability of all tested genes in both tissues indicating that b-actin, B2M, GAPDH and HPRT are appropriate reference genes for the sleep-related gene expression studies. In order to further verify these results, the relative expression of brain derived neurotrophic factor (BDNF) and glycerol-3-phosphate dehydrogenase1 (GPD1) was evaluated in brain and blood, respectively. The normalization with each of four reference genes produced similar pattern of expression in control and sleep deprived rats, but subtle differences in the magnitude of expression fold change were observed which might affect the statistical significance. CONCLUSION: This study demonstrated that sleep deprivation does not alter the expression stability of commonly used reference genes in brain and blood. Nonetheless, the use of multiple reference genes in quantitative RT-PCR is required for the accurate results.

Transduced human PEP-1-heat shock protein 27 efficiently protects against brain ischemic insult.

Reactive oxygen species contribute to the development of various human diseases. Ischemia is characterized by both significant oxidative stress and characteristic changes in the antioxidant defense mechanism. Heat shock protein 27 (HSP27) has a potent ability to increase cell survival in response to oxidative stress. In the present study, we have investigated the protective effects of PEP-1-HSP27 against cell death and ischemic insults. When PEP-1-HSP27 fusion protein was added to the culture medium of astrocyte and primary neuronal cells, it rapidly entered the cells and protected them against cell death induced by oxidative stress. Immunohistochemical analysis revealed that, when PEP-1-HSP27 fusion protein was intraperitoneally injected into gerbils, it prevented neuronal cell death in the CA1 region of the hippocampus in response to transient forebrain ischemia. Our results demonstrate that transduced PEP-1-HSP27 protects against cell death in vitro and in vivo, and suggest that transduction of PEP-1-HSP27 fusion protein provides a potential strategy for therapeutic delivery in various human diseases in which reactive oxygen species are implicated, including stroke.

Hemin interactions and alterations of the subcellular localization of prion protein.

Hemin (iron protoporphyrin IX) is a crucial component of many physiological processes acting either as a prosthetic group or as an intracellular messenger. Some unnatural, synthetic porphyrins have potent anti-scrapie activity and can interact with normal prion protein (PrPC). These observations raised the possibility that hemin, as a natural porphyrin, is a physiological ligand for PrPC. Accordingly, we evaluated PrPC interactions with hemin. When hemin (3-10 microM) was added to the medium of cultured cells, clusters of PrPC formed on the cell surface, and the detergent solubility of PrPC decreased. The addition of hemin also induced PrPC internalization and turnover. The ability of hemin to bind directly to PrPC was demonstrated by hemin-agarose affinity chromatography and UV-visible spectroscopy. Multiple hemin molecules bound primarily to the N-terminal third of PrPC, with reduced binding to PrPC lacking residues 34-94. These hemin-PrPC interactions suggest that PrPC may participate in hemin homeostasis, sensing, and/or uptake and that hemin might affect PrPC functions.

Stress-inducible protein 1 is a cell surface ligand for cellular prion that triggers neuroprotection.

Prions are composed of an isoform of a normal sialoglycoprotein called PrP(c), whose physiological role has been under investigation, with focus on the screening for ligands. Our group described a membrane 66 kDa PrP(c)-binding protein with the aid of antibodies against a peptide deduced by complementary hydropathy. Using these antibodies in western blots from two-dimensional protein gels followed by sequencing the specific spot, we have now identified the molecule as stress-inducible protein 1 (STI1). We show that this protein is also found at the cell membrane besides the cytoplasm. Both proteins interact in a specific and high affinity manner with a K(d) of 10(-7) M. The interaction sites were mapped to amino acids 113-128 from PrP(c) and 230-245 from STI1. Cell surface binding and pull-down experiments showed that recombinant PrP(c) binds to cellular STI1, and co-immunoprecipitation assays strongly suggest that both proteins are associated in vivo. Moreover, PrP(c) interaction with either STI1 or with the peptide we found that represents the binding domain in STI1 induce neuroprotective signals that rescue cells from apoptosis.

Endocytic intermediates involved with the intracellular trafficking of a fluorescent cellular prion protein.

We have investigated the intracellular traffic of PrP(c), a glycosylphosphatidylinositol (GPI)-anchored protein implicated in spongiform encephalopathies. A fluorescent functional green fluorescent protein (GFP)-tagged version of PrP(c) is found at the cell surface and in intracellular compartments in SN56 cells. Confocal microscopy and organelle-specific markers suggest that the protein is found in both the Golgi and the recycling endosomal compartment. Perturbation of endocytosis with a dynamin I-K44A dominant-negative mutant altered the steady-state distribution of the GFP-PrP(c), leading to the accumulation of fluorescence in unfissioned endocytic intermediates. These pre-endocytic intermediates did not seem to accumulate GFP-GPI, a minimum GPI-anchored protein, suggesting that PrP(c) trafficking does not depend solely on the GPI anchor. We found that internalized GFP-PrP(c) accumulates in Rab5-positive endosomes and that a Rab5 mutant alters the steady-state distribution of GFP-PrP(c) but not that of GFP-GPI between the plasma membrane and early endosomes. Therefore, we conclude that PrP(c) internalizes via a dynamin-dependent endocytic pathway and that the protein is targeted to the recycling endosomal compartment via Rab5-positive early endosomes. These observations indicate that traffic of GFP-PrP(c) is not determined predominantly by the GPI anchor and that, different from other GPI-anchored proteins, PrP(c) is delivered to classic endosomes after internalization.

Regulation of the cellular prion protein gene expression depends on chromatin conformation.

Conversion of the normal cellular prion protein (PrPc), whose physiological function is still under investigation, to an infectious form called prion is the cause of some neurodegenerative diseases. Therefore, the elucidation of PrPc gene regulation is important both to define a strategy to control the infection and to better understand PrPc function. We cloned the rat PrPc gene promoter region into a luciferase reporter vector, transfected C6 and PC-12 cells, and isolated clones with stable enzyme expression. The dependence of chromatin conformation on PrPc promoter activity was evaluated using the histone deacetylase inhibitor, trichostatin A, which was able to highly increase not only promoter activity but also PrPc mRNA and protein levels. The phorbol ester (12-O-tetradecanoylphorbol-13-acetate) and cAMP poorly induced promoter activity; retinoic acid decreased it by 50%, whereas nerve growth factor and dexamethasone had no effect. When 12-O-tetradecanoylphorbol-13-acetate or cAMP but not retinoic acid was associated with trichostatin A, a potentiation of the primary effects was observed. These new data indicate that PrPc gene regulation is highly dependent on disruption of chromatin fiber assembly, which allows some ubiquitous transcription factors accession to specific DNA elements.