A role for foregut tyrosine metabolism in glucose tolerance

Objective: We hypothesized that DA and L-DOPA derived from nutritional tyrosine and the resultant observed postprandial plasma excursions of L-DOPA and DA might affect glucose tolerance via their ability to be taken-up by beta cells and inhibit glucose-stimulated b-cell insulin secretion. Methods: To investigate a possible circuit between meal-stimulated 3,4-dihydroxy-L-phenylalanine (L-DOPA) and dopamine (DA) production in the GI tract and pancreatic b-cells, we: 1) mapped GI mucosal expression of tyrosine hydroxylase (TH) and aromatic amino acid decarboxylase (AADC); 2) measured L-DOPA and DA content of GI mucosal tissues following meal challenges with different L-tyrosine (TYR) content, 3) determined whether meal TYR content impacts plasma insulin and glucose excursions; and 4) characterized postprandial plasma excursions of L-DOPA and DA in response to meal tyrosine content in rodents and a population of bariatric surgery patients. Next, we characterized: 1) the metabolic transformation of TYR and L-DOPA into DA in vitro using purified islet tissue; 2) the metabolic transformation of orally administrated stable isotope labeled TYR into pancreatic DA, and 3) using a nuclear medicine technique, we studied endocrine beta cells in situ release and binding of DA in response to a glucose challenge. Results: We demonstrate in rodents that intestinal content and circulatory concentrations L-DOPA and DA, plasma glucose and insulin are responsive to the tyrosine (TYR) content of a test meal. Intestinal expression of two enzymes, Tyrosine hydroxylase (TH) and Aromatic Amino acid Decarboxylase (AADC), essential to the transformation of TYR to DA was mapped and the metabolism of metabolism of TYR to DA was traced in human islets and a rodent beta cell line in vitro and from gut to the pancreas in vivo. Lastly, we show that b cells secrete and bind DA in situ in response to glucose stimulation. Conclusions: We provide proof-of-principle evidence for the existence of a novel postprandial circuit of glucose homeostasis dependent on nutritional tyrosine. DA and L-DOPA derived from nutritional tyrosine may serve to defend against hypoglycemia via inhibition of glucosestimulated b-cell insulin secretion as proposed by the anti-incretin hypothesis

Characterisation of the Haemodynamic Response Function (HRF) in the neonatal brain using functional MRI

Background: Preterm birth is associated with a marked increase in the risk of later neurodevelopmental impairment. With the incidence rising, novel tools are needed to provide an improved understanding of the underlying pathology and better prognostic information. Functional Magnetic Resonance Imaging (fMRI) with Blood Oxygen Level Dependent (BOLD) contrast has the potential to add greatly to the knowledge gained through traditional MRI techniques. However, it has been rarely used with neonatal subjects due to difficulties in application and inconsistent results. Central to this is uncertainity regarding the effects of early brain development on the Haemodynamic Response Function (HRF), knowledge of which is fundamental to fMRI methodology and analysis. Hypotheses: (1) Well localised and positive BOLD functional responses can be identified in the neonatal brain. (2) The morphology of the neonatal HRF differs significantly during early human development. (3) The application of an age-appropriate HRF will improve the identification of functional responses in neonatal fMRI studies. Methods: To test these hypotheses, a systematic fMRI study of neonatal subjects was carried out using a custom made somatosensory stimulus, and an adapted study design and analysis pipeline. The neonatal HRF was then characterised using an event related study design. The potential future application of the findings was then tested in a series of small experiments. Results: Well localised and positive BOLD functional responses were identified in neonatal subjects, with a maturational tendency towards an increasingly complex pattern of activation. A positive amplitude HRF was identified in neonatal subjects, with a maturational trend of a decreasing time-to-peak and increasing positive peak amplitude. Application of the empirical HRF significantly improved the precision of analysis in further fMRI studies. Conclusions: fMRI can be used to study functional activity in the neonatal brain, and may provide vital new information about both development and pathology

Brain opioid and endocannabinoid systems as risk factors for obesity. Positron emission tomography studies of μ-opioid and CB1 receptors with glucose uptake analysis

The prevalence of obesity is increasing globally. Obesity is a major threat to public health since it predisposes individuals to multiple non-communicable diseases. Obesity is difficult to treat or prevent. The modern environment has been blamed for the obesity epidemic, due to the abundance of energy-dense and aggressively advertised foods. The brain is the most important organ controlling energy homeostasis and feeding. However, we do not know which brain pathways render some individuals susceptible to the obesity development in the current environment. The aim of this thesis was to examine whether variation in the brain opioid and endocannabinoid pathways explains differences in the risk for obesity development. Two receptor systems associated with food intake and reward processing were investigated: μ-opioid receptors (MOR) and cannabinoid CB1 receptors (CB1R). MORs were measured with [11C]carfentanil, and CB1Rs with [18F]FMPEP-d2. In addition, brain glucose uptake (BGU) was quantified with [18F]FDG. Healthy, non-obese humans were studied with positron emission tomography in four studies investigating I) the effects of demographic factors on MORs, II) the associations of obesity risk factors on MORs, CB1Rs and BGU, III) the physical fitness and MOR function, and IV) how MORs and CB1Rs associate with feeding behavior. Age, sex and smoking influenced MOR availability, which may contribute to obesity development in specific populations. Familial obesity risk associated with increased BGU but low neuroreceptor availability, suggesting that vulnerability to obesity may be mediated by disruption of these interconnected pathways. Impulsive feeding was associated with reduced MOR availability, which may underlie excessive food intake and weight gain. Central capacity for releasing endogenous MOR ligands was dependent on aerobic fitness, suggesting that the MOR function may be critical in habitual exercise and weight maintenance. Obesity risk factors and circulating cannabinoids associated with reduced CB1R availability, suggesting that an overactive cannabinoid system may facilitate weight gain. In conclusion, multiple neurochemical alterations previously associated with obesity are already present in a number of non-obese individuals, which may increase their risk for future obesity.Aivojen opioidi- ja endokannabinoidijärjestelmät lihavuuden riskitekijöinä. Positroniemissiotomografiatutkimuksia μ-opioidi- ja CB1-reseptoreista sekä glukoosin otosta Lihavuus yleistyy ympäri maailmaa. Lihavuus on yksi merkittävimmistä uhista väestön terveydelle, sillä painon kertyminen altistaa useille kansansairauksille. Lihavuutta on vaikea hoitaa tai ehkäistä. Nykyistä elinympäristöä on syytetty lihavuusepidemiasta, sillä ympäristö on täynnä energiatiiviitä ja voimakkaasti markkinoituja ruokatuotteita. Aivot ovat tärkein energiatasapainoa ja syömistä säätelevä elin. Emme kuitenkaan tiedä, mitkä muutokset aivojen toiminnassa saavat osan ihmisistä lihomaan tässä ympäristössä. Väitöskirjan tavoitteena oli selvittää, selittävätkö aivojen opioidi- ja endokannabinoidijärjestelmän muutokset eroja ihmisten välisessä lihomisriskissä. Tutkimme kahta aivojen välittäjäainejärjestelmää, jotka säätelevät syömisen palkkiokokemuksia: μ-opioidireseptoreja (MOR) ja CB1-kannabinoidireseptoreja (CB1R). MOR-sitoutumista mitattiin [11C]karfentaniililla, ja CB1R-sitoutumista [18F]FMPEP-d2-merkkaineella. Aivojen glukoosinottoa mitattiin lisäksi [18F]FDG-merkkiaineella. Tutkimme terveitä, ei-lihavia ihmisiä positroniemissiotomografialla neljässä tutkimuksessa, joissa selvitettiin: I) väestömuuttujien vaikutusta MOR-sitoutumiseen, II) lihavuuden riskitekijöiden vaikutusta MOR- ja CB1R-sitoutumiseen sekä aivojen glukoosinottoon, III) fyysistä kuntoa ja MOR-toimintaa, ja IV) MOR- ja CB1R-sitoitumisen yhteyttä syömiskäyttäytymiseen. Ikä, sukupuoli ja tupakointi vaikuttivat MOR-sitoutumiseen, mikä voi selittää eroja lihavuuden kehittymisessä eri väestöryhmissä. Perheeseen liittyvä lihomisriski oli yhteydessä aivojen glukoosinottoon ja reseptorimääriin, ja näiden järjestelmien häiriintyminen saattaa altistaa lihavuudelle. Impulsiivinen syömiskäyttäytyminen liittyi alentuneeseen MOR-sitoutumiseen, mikä voi altistaa liialliselle syömiselle ja painon nousulle. Sisäsyntyisten opioidien vapauttamiskyky oli yhteydessä fyysiseen kuntoon, viitaten MOR-toiminnan merkitykseen liikuntaharrastuksen ylläpidossa ja painonhallinnassa. Lihavuuden riskitekijät ja verenkierron kannabinoidit liittyivät alentuneeseen CB1R-sitoutumiseen, mikä viittaa siihen, että yliaktiivinen kannabinoidijärjestelmä voi altistaa lihomiselle. Osalla terveistä ihmisistä on siis havaittavissa useita aivokemiallisia muutoksia, jotka saattavat altistaa lihavuudelle

Kynurenines in neurological disorders

The kynurenine pathway is thought to be involved in neurological disorders but its precise role and the mechanisms involved have yet to be established. Tryptophan can be metabolised via this pathway to produce the neurotoxic N-methyl-D-aspartate (NMDA) receptor agonist, quinolinic acid (QUIN), and the direct generators of reactive oxygen species, 3-hydroxykynurenine (3HKYN) and 3-hydroxyanthranilic acid (3HANA), as well as the neuroprotective NMDA receptor antagonist, kynurenic acid (KYNA). High performance liquid chromatography (HPLC) methods were successfully developed and validated for measuring tryptophan, kynurenine, KYNA, 3HANA and anthranilic acid (ANA) in blood samples, using absorbance and fluorescence detection. The method for determining 3HKYN using electrochemical detection was more problematic and was only used for tryptophan loaded samples and their respective baseline samples. Using HPLC, the concentrations of tryptophan, kynurenine, KYNA, 3HKYN and 3HANA were measured in the blood of Huntington's disease (HD) patients and patients with chronic brain injury, where the injury had occurred at least one year previously. QUIN was also determined for these patients using gas chromatography-mass spectrometry (GC-MS). In addition, the dynamics of the kynurenine pathway were investigated following oral tryptophan depletion and loading. In contrast to these chronic conditions, patients with acute stroke were also studied. The concentrations of tryptophan, kynurenine, KYNA, ANA and 3HANA were determined in the blood of the stroke patients, examining any changes in these concentrations during the two weeks after the stroke. The extent of inflammation and oxidative stress were also assessed for all patients, by measuring the levels of neopterin and the lipid peroxidation products, malondialdehyde and 4-hydroxynonenal. Patients with late stage HD showed abnormal tryptophan metabolism via the kynurenine pathway, together with increased inflammation and oxidative stress. Increased levels of kynurenine together with increased kynurenine: tryptophan (K:T) ratios, indicating greater indoleamine 2,3-dioxygenase (IDO) activity, were observed in blood samples from HD patients in comparison with healthy control subjects. In conjunction with this increased IDO activity, there was a decrease in the ratios of KYNA: kynurenine, suggesting decreased kynurenine aminotransferase (KAT) activity. Inflammation, which may be stimulating IDO activity, could also be decreasing KAT activity, suggested by negativecorrelations between the KYNA: kynurenine ratios and the inflammatory marker, neopterin. The inactivity of KAT suggests a small deficiency in KYNA over a long period of time which could cause a reduction in NMDA receptor antagonism, resulting in slow progressive excitotoxicity contributing to the neurodegeneration in HD. Low KYNA: kynurenine ratios were observed in baseline and tryptophan depleted samples, but after tryptophan loading, HD patients showed similar ratios compared with control subjects. This suggests that loading may be beneficial for HD patients, as more of the neuroprotectant, KYNA can potentially be produced. However, the results suggest that concentrations of the neurotoxin, QUIN, may also be increasing after tryptophan loading. Low concentrations of 3HKYN and 3HANA, with no change in QUIN levels, were also observed in the blood of HD patients. 3HANA levels continued to be decreased for the HD patients after loading. This may suggest degradation of 3HKYN and 3HANA by autoxidation producing reactive oxygen species which could contribute to the high levels of oxidative stress found in these patients. Tryptophan loading in healthy control subjects showed significant increases in the inflammatory marker, neopterin, and in the lipid peroxidation products. These results should be considered when tryptophan loading is used in psychiatric practice and in diets high in tryptophan, such as the Atkins diet. Patients with severe chronic brain injury showed similar alterations in kynurenine pathway metabolism as HD patients, at baseline and throughout the loading and depletion protocols. Although the brain injury had occurred at least one year previously, these patients showed persistent inflammation and oxidative stress, demonstrated by their increased levels of neopterin and lipid peroxidation products compared with healthy controls. In baseline blood samples, there were increased K:T ratios indicating greater IDO activity in the patients. Patients with chronic brain injury showed decreased concentrations of the neuroprotectant, KYNA, as well as low KAT activity, indicated by the decreased KYNA: kynurenine ratios. After tryptophan loading, K:T ratios decreased and the KYNA: kynurenine ratios increased in patients in comparison with controls, suggesting a reversal in the activities of the enzymes IDO and KAT. Similar levels of the inflammatory marker, neopterin, were observed in patients and controls after tryptophan loading. This suggests that these changes in IDO and KAT activities may be related to inflammation. As for the HD patients, patients with chronic brain injury showed lower levels of 3HKYN and 3HANA in their blood, with no change in QUIN levels. These metabolites may be undergoing autoxidation, producing reactive oxygen species which contribute to the ongoing oxidative stress in these patients.The kynurenine pathway was activated following an acute stroke, as indicated by the increased K:T ratios, suggesting greater IDO activity. Stroke patients also had raised levels of neopterin and lipid peroxidation products, indicating inflammation and oxidative stress. There were no changes in the blood concentrations of kynurenines, neopterin or lipid peroxidation products during the fourteen days after a stroke. Stroke patients had reduced levels of 3HANA in their blood, as observed for the HD and chronic brain injury patients. There were negative correlations between the concentration of 3HANA and the volume of the brain lesion, measured by computed tomography (CT) scan, demonstrating the importance of the decreased concentrations of 3HANA. In addition, there were increased levels of ANA in the blood of the stroke patients and the ratios of 3HANA: ANA also correlated with brain lesion volume. Another measurement which correlated with lesion volume was lipid peroxidation, suggesting that oxidative stress contributes to the extent of the brain damage after a stroke. This may suggest that the role of 3HANA in stroke is related to its autoxidation and the generation of reactive oxygen species. Increased concentrations of KYNA were observed in patients who died within three weeks of having a stroke. These high levels of KYNA may have been produced following excitotoxicity and the generation of free radicals, and may cause excessive NMDA receptor blockade or reduced mitochondrial adenosine triphosphate (ATP) synthesis, thus contributing to cell death. The kynurenine pathway was activated and showed abnormal metabolism in all the patient groups, suggesting a potential role for these metabolites in neuronal dysfunction in HD, chronic brain injury and acute stroke. Further work is required to elucidate the role of tryptophan metabolites and whether they may have a direct contribution to neuronal damage in neurological disorders

Doctor of Philosophy

dissertationDepressive disorders (DD) are a leading cause of disability worldwide and display diverse symptoms including anhedonia, melancholia, decreased concentration, sleep and appetite disturbances, and suicidal thoughts and acts. Current available medications are still ineffective for more than 30% of patients, suggesting DD are multi-faceted heterogeneous disorders. Despite intense research, as yet there are no widely used biological diagnostic tests for DD. Since DD are likely a manifestation of both genetic and environmental factors, gene expression of peripheral blood leukocyte allows for a non-invasive means to evaluate trait- and state-dependent neurophysiological dysregulation. In this dissertation, we employed real-time quantitative polymerase chain reaction (qPCR) to evaluate differences between healthy controls and patients with medication-refractory depression, for a panel of candidate genes previously implicated in depression as well as novel genes implicated in related neurological disorders. Chapter 1 begins with an overview of the multiple domains involved in depression and of previous literature findings evaluating protein and gene expression. Chapter 2 describes one of our studies of gene expression of a small panel of 14 genes involved in the immune and stress response in 19 patients with medication-refractory depression, before and following symptom improvement, compared to 20 healthy controls. We found that patients displayed trait- and state-dependent dysregulation in immune cytokines IL-10 and IL-6, transcription factor NFkB1, the serotonin receptor HTR1D, GABAA modulator DBI, and the adrenergic receptors ADR2A and ADRB1. Furthermore, the dopamine receptor DRD4, the glucocorticoid receptor NR3C1, and SULT1A1 displayed acute changes following treatment, though no differences were observed Pretreatment. In Chapter 3, we describe another gene expression study with results using a larger sample size (42 patients and 38 controls) and an expanded gene panel (46 genes) that includes candidate genes from diverse biological pathways. In this study, we found upregulation of IL-10 and IL-6 as well as dysregulation in the amyloid precursor protein, neuregulin-1, and several ion channels that have roles in anxiety, pain, and fatigue. Finally, Chapter 4 summarizes results from both studies and discusses future research into promising biomarkers and novel mechanisms of depression

Alterations in circadian entrainment : translational value in major depression

Adverse conditions of different origin occurring during development can have a negative impact on the nervous system and increase risk for the onset of mental disorders. In mice prenatal exposure to excess glucocorticoids (GC) leads to late onset depression-like behaviour in males which is preceded by alterations in circadian entrainment. The aims of this thesis were to (1) identify sex-related differences in behavioural outcomes with a special focus on circadian activity and mood-related behaviours; (2) investigate the mechanisms behind the behavioural alterations; (3) implement novel computational techniques for characterisation of behaviour in rodents; and (4) explore the translation potential of the analysis of activity in patients suffering from depression. Pregnant C57Bl/6 were exposed to the synthetic GC dexamethasone (Dex) (0.05 mg/Kg/day, i.p.) from gestational day 14 until delivery. We analysed the behaviour of both males and female offspring at the age of 6 months focusing on circadian entrainment of spontaneous activity in the home cage environment. There were sparse differences between Dex and control (Ctrls) males at baseline, which were consistent with decreased social behaviour. In contrast, Dex-females were spontaneously hyperactive, a phenotype resembling ADHD models. Next, we investigated the changes in spontaneous activity following an abrupt advance in dark phase onset (phase shift). Dex-males re-entrained activity faster, while in Dex-females re- entrainment was delayed as compared to Ctrls. We implemented a multivariate approach to analysing the spatial and temporal organisation of behaviour which included affinity propagation (AP) clustering and uniform manifold approximation and projection (UMAP) using a set of 129 features extracted form activity recording. AP-clustering allowed the identification of features with similar patterns of variations following the phase shift, while UMAP supported the visualisation and quantification of changes in organisation of behaviour. The organisation of behaviour changed in both males and females, particularly in the dark phase. Dex-males displayed fewer, but more persistent alterations, while in Dex- females most alterations were transient and returned to baseline values within 5 days after the phase shift. The behaviour alterations in response to the phase shift pointed at alterations in the function of the central clock, the suprachiasmatic nucleus (SCN). In Dex-males the coupling between SCN and peripheral oscillators was disrupted, but was restored by enhancing GC receptor-mediated signalling by the antidepressant DMI. Furthermore, DMI also prevented the onset of depression. In Dex-females instead, the coupling between SCN and peripheral oscillators was preserved, supporting the idea that altered coupling is an early sign specific for depression onset. To investigate the connection between Dex-female behaviour and central clock alterations we performed gene expression analysis by RNA- sequencing followed by SPIA pathway analysis and identified significant alterations in glutamatergic, GABAergic, and dopaminergic signalling. We selected a set of relevant genes to validate the altered expression by cross-referencing differentially expressed genes in the SCN with the international mouse phenotype consortium (IMPC) database for associated phenotypes. Using this approach, we identified several differentially regulated genes supproting a decrease in dopamine signalling and the ADHD-like phenotype in Dex-females. Spontaneous activity can be measured also in human subjects by means of actigraphy. We used actigraphy recordings from patients suffering from major depression to explore possible correlations between patterns of activity and symptom severity or response to internet- delivered cognitive behavioural therapy (iCBT) as antidepressant treatment. We implemented an independent homogenous training of model ensembles using systematic bootstrapping (with replacement) followed by pruning based on goodness-of-fit criteria. The aggregated output outperformed individual models by a factor of 4, indicating that ensemble training increases the accuracy of fitting. We further applied an external validation procedure using an independent dataset to provide proof-of-concept evidence for the possibility to model symptom severity estimated by MADRS-S. Our data suggests that higher symptom severity is associated with less complex patterns of activity, stronger coupling with circadian entrainers and less robust circadian rhythms, while larger improvement following iCBT is associated with less fragmented activity, more robust circadian rhythms, and higher day-to- day variability before treatment. To summarize, these studies suggest a strong connection between circadian activity and neuropsychiatric disorders. Further experimental and clinical investigations are needed to dissect all relevant systems and molecules involved to provide new insight into mental disorders

Detecting diverse types of cardiovascular brain pulses in Alzheimer’s disease simultaneously with fNIRS and MREG

Abstract. One of the risk factors for Alzheimer’s disease is hypertension. Hypertension alters the brain’s blood vessel structure due to increased arterial pressure. Structural changes in the blood vessels are seen in the cardiovascular pulse, which is formed by blood velocity, blood flow rate, blood pressure, and infrequently blood flow. By simultaneously applying magnetic resonance encephalography (MREG) and functional near-infrared spectroscopy (fNIRS), this study discovered cardiovascular brain pulses from the blood flow within patients with Alzheimer’s disease and healthy controls. This study detects specific parameters within diverse types of cardiovascular brain pulses. The results detected changes in parameters for diverse types of cardiovascular brain pulses in patients with Alzheimer’s disease within MREG and fNIRS. In addition, the results present an alternative method for finding cardiovascular brain pulse from the blood flow, which might reflect the structural changes of a blood vessel in patients with Alzheimer’s disease. In conclusion, diverse types of cardiovascular brain pulses represent an approximation of arterial, venous, and tissue pulses, which is beneficial for distinguishing the effect of venous and arterial hypertension in Alzheimer’s disease. Furthermore, altered blood flow may potentially be associated with the impaired glymphatic system in Alzheimer’s disease

Development of Low-Frequency Repetitive Transcranial Magnetic Stimulation as a Tool to Modulate Visual Disorders: Insights from Neuroimaging

Repetitive transcranial magnetic stimulation (rTMS) has become a popular neuromodulation technique, increasingly employed to manage several neurological and psychological conditions. Despite its popular use, the underlying mechanisms of rTMS remain largely unknown, particularly at the visual cortex. Moreover, the application of rTMS to modulate visual-related disorders is under-investigated. The goal of the present research was to address these issues. I employ a multitude of neuroimaging techniques to gain further insight into neural mechanisms underlying low-frequency (1 Hz) rTMS to the visual cortex. In addition, I begin to develop and refine clinical low-frequency rTMS protocols applicable to visual disorders as an alternative therapy where other treatment options are unsuccessful or where there are simply no existing therapies. One such visual disorder that can benefit from rTMS treatment is the perception of visual hallucinations that can occur following visual pathway damage in otherwise cognitively healthy individuals. In Chapters 23, I investigate the potential of multiday low-frequency rTMS to the visual cortex to alleviate continuous and disruptive visual hallucinations consequent to occipital injury. Combining rTMS with magnetic resonance imaging techniques reveals functional and structural cortical changes that lead to the perception of visual hallucinations; and rTMS successfully attenuates these anomalous visual perceptions. In Chapters 45, I compare the effects of alternative doses of low-frequency rTMS to the visual cortex on neurotransmitter levels and intrinsic functional connectivity to gain insight into rTMS mechanisms and establish the most effective protocol. Differential dose-dependent effects are observed on neurotransmitter levels and functional connectivity that suggest the choice of protocol critically depends on the neurophysiological target. Collectively, this work provides a basic framework for the use of low-frequency rTMS and neuroimaging in clinical application for visual disorders

Interpreting EEG alpha activity

Exploring EEG alpha oscillations has generated considerable interest, in particular with regards to the role they play in cognitive, psychomotor, psycho-emotional and physiological aspects of human life. However, there is no clearly agreed upon definition of what constitutes ‘alpha activity’ or which of the many indices should be used to characterize it. To address these issues this review attempts to delineate EEG alpha-activity, its physical, molecular and morphological nature, and examine the following indices: (1) the individual alpha peak frequency; (2) activation magnitude, as measured by alpha amplitude suppression across the individual alpha bandwidth in response to eyes opening, and (3) alpha “auto-rhythmicity” indices: which include intra-spindle amplitude variability, spindle length and steepness. Throughout, the article offers a number of suggestions regarding the mechanism(s) of alpha activity related to inter and intra-individual variability. In addition, it provides some insights into the various psychophysiological indices of alpha activity and highlights their role in optimal functioning and behavior