Peter Brust

Adenosine activates brown adipose tissue and recruits beige adipocytes via A2A receptors

Brown adipose tissue (BAT) is specialized in energy expenditure, making it a potential target for anti-obesity therapies. Following exposure to cold, BAT is activated by the sympathetic nervous system with concomitant release of catecholamines and activation of β-adrenergic receptors. Because BAT therapies based on cold exposure or β-adrenergic agonists are clinically not feasible, alternative strategies must be explored. Purinergic co-transmission might be involved in sympathetic control of BAT and previous studies reported inhibitory effects of the purinergic transmitter adenosine in BAT from hamster or rat. However, the role of adenosine in human BAT is unknown. Here we show that adenosine activates human and murine brown adipocytes at low nanomolar concentrations. Adenosine is released in BAT during stimulation of sympathetic nerves as well as from brown adipocytes. The adenosine A2A receptor is the most abundant adenosine receptor in human and murine BAT. Pharmacological blockade or genetic loss of A2A receptors in mice causes a decrease in BAT-dependent thermogenesis, whereas treatment with A2A agonists significantly increases energy expenditure. Moreover, pharmacological stimulation of A2A receptors or injection of lentiviral vectors expressing the A2A receptor into white fat induces brown-like cells—so-called beige adipocytes. Importantly, mice fed a high-fat diet and treated with an A2A agonist are leaner with improved glucose tolerance. Taken together, our results demonstrate that adenosine–A2A signalling plays an unexpected physiological role in sympathetic BAT activation and protects mice from diet-induced obesity. Those findings reveal new possibilities for developing novel obesity therapies.

Reduced α4β2*–Nicotinic Acetylcholine Receptor Binding and Its Relationship to Mild Cognitive and Depressive Symptoms in Parkinson Disease

CONTEXT Cognitive or depressive disorders are frequently noted in patients with Parkinson disease (PD) and may be related to altered signaling through alpha4beta2*-nicotinic acetylcholine receptors (alpha4beta2*-nAChRs). OBJECTIVE To assess the availability of alpha4beta2*-nAChRs and their relationship to mild cognitive and mild depressive symptoms in vivo in patients with PD. DESIGN Crossover comparison between patients with PD and healthy volunteers (control group) using the alpha4beta2*-nAChR-specific radioligand 2-[(18)F]fluoro-3-(2[S]-2-azetidinylmethoxy)-pyridine (2-[(18)F]FA-85380) and positron emission tomography. SETTING Departments of Neurology and Nuclear Medicine, University of Leipzig, Leipzig, Germany. PARTICIPANTS Twenty-two nonsmoking patients with PD and 9 nonsmoking healthy volunteers. MAIN OUTCOME MEASURES Level of 2-[(18)F]FA-85380 binding potential (2-FA BP), a measure of alpha4beta2*-nAChR availability. The relationship between severity of cognitive symptoms as rated using the Mini-Mental State Examination and DemTect scale and the level of depressive symptoms as indicated using the Beck Depression Inventory, and 2-FA BP were assessed. RESULTS In patients with PD compared with healthy volunteers, there was widespread reduced 2-FA BP, especially in the midbrain, pons, anterior cingulate cortex, frontoparietal cortex, and cerebellum. In subgroups of patients with PD with possible depression, reduced 2-FA BP was most pronounced in the cingulate cortex and frontoparieto-occipital cortex, whereas in patients with PD with mild cognitive impairment, 2-FA BP was reduced in the midbrain, pons, and cerebellum. In patients with PD, the strongest associations between depressive symptoms and reduced 2-FA BP were noted in the anterior cingulate cortex, putamen, midbrain, and occipital cortex. In contrast, cognitive symptoms correlated only weakly with reduced 2-FA BP in the thalamus, midbrain, temporal cortex, hippocampus, and cerebellum. CONCLUSIONS There is a broad reduction of alpha4beta2*-nAChR availability in patients with PD without clinically manifest dementia or depression compared with healthy volunteers. Reduced alpha4beta2*-nAChR binding in patients with PD within the subcortical and cortical regions is associated with the severity of mild cognitive or depressive symptoms. These results provide novel in vivo evidence for a role of the cholinergic neurotransmission in psychiatric comorbidity of PD.

Acetylcholine receptors in dementia and mild cognitive impairment

PurposeTo clarify whether changes in the cholinergic transmission occur early in the course of Alzheimer’s disease (AD), we carried out positron emission tomography (PET) with the radioligand 2-[18F]F-A-85380, which is supposed to be specific for α4β2 nicotinic acetylcholine receptors (nAChRs).MethodWe included patients with moderate to severe AD and patients with amnestic mild cognitive impairment (MCI), presumed to present preclinical AD.ResultsBoth patients with AD and MCI showed significant reductions in α4β2 nAChRs in brain regions typically affected by AD pathology. These findings indicate that a reduction in α4β2 nAChRs occurs during early symptomatic stages of AD. The α4β2 nAChR availability in these regions correlated with the severity of cognitive impairment, indicating a stage sensitivity of the α4β2 nAChR status.ConclusionTogether, our results provide evidence for the potential of 2-[18]F-A-85380 nAChR PET in the diagnosis of patients at risk for AD. Because of the extraordinary long acquisition time with 2-[18F]F-A-85380, we developed the new α4β2 nAChR-specific radioligands (+)- and (−)-[18F]norchloro-fluoro-homoepibatidine (NCFHEB) and evaluated them preclinically. (−)-[18F]NCFHEB shows twofold higher brain uptake and significantly shorter acquisition times. Therefore, (−)-[18F]NCFHEB should be a suitable radioligand for larger clinical investigations.

Impact of asymmetric intrauterine growth restriction on organ function in newborn piglets

Fetal malnutrition may induce asymmetric intrauterine growth restriction (aIUGR) with long-lasting consequences. Understanding the organ-specific structural and functional effects aIUGR may have on the newborn, and understanding the potential impact on the neonatal response to compromising conditions, appears to be essential for adequate treatment. Therefore, a survey is given of some organ-specific alterations in newborns, which have suffered from aIUGR. We studied these effects in a model of asymmetric intrauterine growth restriction based on the spontaneous occurrence of runting in pigs. We wish to demonstrate that experimental studies in animal models are necessary and helpful to elucidate pathogenetic mechanisms. aIUGR seems to have both beneficial and detrimental effects on the newborn. The development of skeletal muscles (conversion to oxidative type I fibers) and of their vascular supply as well as of the brain dopaminergic activity is accelerated. Also, aIUGR apparently improves the ability to withstand critical periods of gradual oxygen deficit as shown by the maintenance of renal blood flow during severe systemic hypoxia, and by improved cerebrovascular autoregulation in hemorrhagic hypotension. On the other hand, aIUGR leads to the reduction of the number of nephrons and to impaired renal excretory functions with arterial hypertension and chronic renal failure.

Technetium(v) and rhenium(v) complexes for 5-HT2A serotonin receptor binding: Structure-affinity considerations☆

Starting from the lead structure of ketanserin, a prototypic serotonin (5-HT) antagonist, new oxotechnetium(V) and oxorhenium(V) complexes were synthesized that are able to compete with [3H]ketan-serin in receptor-binding assays. To imitate organic 5-HT2 receptor ligands, fragments of ketanserin were combined with chelate moieties. Neutral compounds of the general formula [MOL1L2] (M = Tc, Re; L1 = HS-CH2CH2-S-CH2CH2-SH, N-(2-mercaptophenyl)salicylideneimine, N-(2-mercaptoethyl)-salicylideneimine, 3-(2-([N,N-bis(2-mercapto-S-ethyl)]-amino)ethyl)-2,4-(1H, 3H)-quinazolinedione and L2 = HS-R with R = subst. alkyl) were prepared by common action of a Tc(V) or Re(V) precursor with a mixture of equimolar amounts of a tridentate ligand L1 and a monodentate thiolate L2 bearing fragments of the lead structure. Lipophilic complexes consisting of a small S4 thiolate/thioether chelate unit, protonable nitrogen-containing spacer, and simple benzyl moiety significantly inhibited the specific binding of [3H]ketan-serin with IC50 values between 10 and 50 nM.

Changes in Regional Blood–Brain Transfer of l-Leucine Elicited by Arginine-Vasopressin

Abstract: Arginine‐vasopressin (AVP), injected into the carotid artery in physiological concentration together with L‐leucine, changed kinetic constants of the blood—brain barrier (BBB) transport of this neutral amino acid without changing the cerebral blood flow (CBF). The maximum velocity of transport (Vmax), the half‐saturation constant (Km), the nonsaturable transport constant (KD), and CBF were estimated in nine brain regions of male Wistar rats anesthetized with ether. In cerebral hemisphere, Vmax decreased from 21 nmol min−1. g−1 (control) to 7.6 nmol. min−1. g−1 (AVP). Km decreased from 0.11 to 0.029 mM. Regional differences of the kinetic constants were found in controls as well as in AVP‐treated animals. In all regions, the calculated constants Vmax and Km of animals coinjected with AVP were significantly decreased when compared to controls. A direct or indirect interaction of AVP with the transport system of large neutral amino acids is suggested.

Functional Expression of the Serotonin Transporter in Immortalized Rat Brain Microvessel Endothelial Cells

There is evidence from recent studies that the brain endothelium (of capillaries and/or larger vessels) may serve as a specific target for serotonin [5‐hydroxytryptamine (5‐HT)]. This neurotransmitter is expected to be involved in the regulation of the blood‐brain barrier (BBB) permeability and/or of the cerebral blood flow via receptor‐mediated mechanisms. Effective control of these processes depends on a speedy uptake and metabolism of released 5‐HT molecules. To realize this, a similar mechanism of 5‐HT uptake as in brain may exist at the BBB. In this study, we have demonstrated using RT‐PCR that 5‐HT transporter mRNA is present in the brain endothelium and that a saturable transport system for 5‐HT is functionally expressed in immortalized rat brain endothelial cells (RBE4 cells). These cells take up [3H]5‐HT by an active saturable process with a Km value of 397 ± 64 nmol/L and a transport capacity of 51.7 ± 3.5 pmol · g‐1· min‐1. The 5‐HT uptake depends on Na+, as indicated by the replacement of NaCl by LiCl. The 5‐HT uptake was sensitive to specific 5‐HT transport inhibitors such as paroxetine, clomipramine, fluoxetine, and citalopram but not to inhibitors of the vesicular amine transporter such as reserpine or tetrabenazine. Our results demonstrate that cerebral endothelial cells are able to participate actively in the removal and metabolism of the released 5‐HT, which supports the concept of direct serotoninergic regulation of the BBB function.

Molecular cloning and functional characterization of the OCTN2 transporter at the RBE4 cells, an in vitro model of the blood–brain barrier

The transport of L-carnitine (4-N-trimethylamino-3-hydroxybutyric acid), a compound known to be transported by the organic cation transporter/carnitine transporter OCTN2, was studied in immortalized rat brain endothelial cells (RBE4). The cells were found to take up L-carnitine by a sodium-dependent process. This uptake process was saturable with an apparent Michaelis-Menten constant for L-carnitine of 54+/-10 microM and a maximal velocity of 215+/-35 pmol/mg protein/h. Besides L-carnitine, the cells also took up acetyl-L-carnitine and propionyl-L-carnitine in a sodium-dependent manner and TEA in a sodium-independent manner. RT-PCR with primers specific for the rat OCTN2 transporter revealed the existence of OCTN2 mRNA in RBE4 cells. Screening of a cDNA library from RBE4 cells with rat OCTN2 cDNA as a probe identified a positive clone which showed, when expressed in HeLa cells, the functional characteristics of OCTN2. The HeLa cells expressing the RBE4 OCTN2 cDNA showed a sixfold increase in L-carnitine uptake and a fourfold increase in TEA uptake in a sodium-containing buffer. Typical inhibitors for organic cation transporters (e.g. MPP(+) or TEA) showed an inhibitory effect on the transport of L-carnitine and TEA into the transfected cells. Similarly, unlabeled L-carnitine inhibited the transport of [3H]-L-carnitine and [14C]TEA in transfected HeLa cells. It is concluded that RBE4 cells, a widely used in vitro model of the blood-brain barrier (BBB), express the organic cation/carnitine transporter OCTN2.

Autoradiographic analyses of 5-HT1A and 5-HT2A receptors after social isolation in mice.

Social isolation of rodents is used to model human psychopathological processes. In the present study, the effects of intermediate and long term isolation housing on postsynaptic 5-HT(1A) and 5-HT(2A) receptors were analyzed in male mice housed in groups or isolation for 4 and 12 weeks. [3H]8-OH-DPAT and [3H]ketanserin were used to label 5-HT(1A) and 5-HT(2A) receptors. Four representative sagittal sections (planes 1-4) were scored by in vitro autoradiography. Whereas after 4 weeks of housing both receptor densities were lowered significantly in isolated mice, after 12 weeks of housing only marginal isolation effects were seen. Intermediate isolation reduced 5-HT(1A) receptors especially in the lateral frontal, parietal and entorhinal cortex (-63%), in the lateral CA1-3 and dentate gyrus region of the hippocampus (-68%), in the basolateral, basomedial, central and medial amygdaloid nuclei (between -38 and -66%), and in the hypothalamus (-28%). 5-HT(2A) receptors were strongly reduced in the frontal cortex (between -47 and -74%), in the hippocampus (between -47 and -95%), in the striatum (between -66 and -76%), and in the accumbens nucleus (between -59 and -73%) in comparison to group housed control mice. After 12 weeks of isolation in the hippocampus continuously decreased 5-HT(1A) receptor densities were demonstrated (between -24 and -61%). But increased 5-HT(2A) receptor densities were seen in the lateral striatum (+86%) compared to control mice. Age-dependent effects were also found. After 12 weeks of group housing the 5-HT(1A) and 5-HT(2A) receptor densities were decreased (between -28 and -54%) in all analyzed brain regions in comparison to 4 weeks of group housing. Isolated animals showed diminished 5-HT(1A) receptor densities in the cortex (-14%) and hippocampus (-15%), but increased 5-HT(1A) receptor densities in the amygdala (+33%) after 12 weeks. The 5-HT(2A) receptor densities were increased in all analyzed regions (between +31 and +96%) after 12 weeks of isolation compared to 4 weeks. To explain these dynamic, time-dependent pattern of isolation-induced changes different regulation processes are supposed regarding 5-HT(1A) and 5-HT(2A) receptors. Besides metabolism-related adaptation processes also neurotransmitter and hormonal (e.g., glucocorticoid) interactions especially in limbic regions have to be considered.

Density of perfused capillaries in living human brain during functional activation

Recent evidence has indicated that functional activation of cerebral cortex is accompanied by increases of blood flow and glucose consumption but not oxygen consumption. No explanation has been advanced for this change of the flow-metabolism couple. We formulated the hypothesis that oxygen delivery to brain tissue is diffusion-limited by the enormous hemoglobin binding, and rate-limiting for the oxygen consumption of the tissue. One prediction of this hypothesis is very low oxygen tensions in the tissue. A second prediction is the inability of oxygen consumption to increase during functional activation in the absence of recruitment of capillaries for the oxygen diffusion capacity. We designed a study to test the latter prediction by calculating the density of functioning capillaries during vibrotactile stimulation of the parietal cortex. We defined functioning capillaries as capillaries that transport glucose and therefore calculated the capillary density from the glucose diffusion capacity (K1) of the cerebral capillaries. We confirmed the presence of a partial flow-CMRglc couple (2:1) during the functional activation. Oxygen consumption did not change despite an increase of capillary density in proportion to the change of blood flow.