Dana S. Hutchinson

Astrocytic involvement in learning and memory consolidation

Astrocytes play fundamental roles in brain function, interacting with neurons and other astrocytes, yet their role in learning is not widely recognized. This review focuses on astrocytic involvement in memory consolidation following bead discrimination learning in day-old chick and draws parallels to mammalian learning, providing strong empirical support for the conclusion that the described neuronal-astrocytic interactions are universally valid. It identifies specific mechanisms whereby astrocytes support memory consolidation. Uptake of glucose, stimulated in astrocytes by beta(3)-noradrenergic receptor activation, provides energy by glycolytic/oxidative metabolism. Unlike neurons, astrocytes carry out net synthesis of tricarboxylic acid cycle intermediates needed for synthesis of transmitter glutamate formed by rapid degradation of glucose-derived glycogen and stimulated by beta(2)-noradrenergic receptor activation. This makes learning dependent on glycogenolysis and its stimulation by noradrenaline. Astrocytes take up most synaptically released glutamate, terminating transmitter activity and returning glutamate to neurons in a glutamate-glutamine cycle, interference with which abolishes learning. The various astrocytic activities follow a rigidly controlled time schedule, easily determined after bead discrimination learning but also detectable in other paradigms.

Ligand‐directed signalling at β‐adrenoceptors

β‐Adrenoceptors (ARs) classically mediate responses to the endogenous ligands adrenaline and noradrenaline by coupling to Gsα and stimulating cAMP production; however, drugs designed as β‐AR agonists or antagonists can activate alternative cell signalling pathways, with the potential to influence clinical efficacy. Furthermore, drugs acting at β‐ARs have differential capacity for pathway activation, described as stimulus trafficking, biased agonism, functional selectivity or ligand‐directed signalling. These terms refer to responses where drug A has higher efficacy than drug B for one signalling pathway, but a lower efficacy than drug B for a second pathway. The accepted explanation for such responses is that drugs A and B have the capacity to induce or stabilize distinct active conformations of the receptor that in turn display altered coupling efficiency to different effectors. This is consistent with biophysical studies showing that drugs can indeed promote distinct conformational states. Agonists acting at β‐ARs display ligand‐directed signalling, but many drugs acting as cAMP antagonists are also able to activate signalling pathways central to cell survival and proliferation or cell death. The observed complexity of drug activity at β‐ARs, prototypical G protein‐coupled receptors, necessitates rethinking of the approaches used for screening and characterization of novel therapeutic agents. Most studies of ligand‐directed signalling employ recombinant cell systems with high receptor abundance. While such systems are valid for examining upstream signalling events, such as receptor conformational changes and G protein activation, they are less robust when comparing downstream signalling outputs as these are likely to be affected by complex pathway interactions.

β-Adrenoceptors, but not α-adrenoceptors, stimulate AMP-activated protein kinase in brown adipocytes independently of uncoupling protein-1

Aims/hypothesisBrown adipocytes provide a potentially important model system for understanding AMP-activated protein kinase (AMPK) regulation, where adrenergic stimulation leads to mitochondrial uncoupling through uncoupling protein-1 (UCP1) activity. AMPK is a sensor of energy homeostasis and has been implicated in glucose and lipid metabolism in several insulin-sensitive tissues. The aim of this study was to characterise the potential role of AMPK in adrenergically mediated glucose uptake and to find out whether UCP1 is involved in the adrenergic activation of AMPK.MethodsWe used primary brown adipocytes differentiated in culture and measured AMPK phosphorylation and glucose uptake following adrenergic activation.ResultsTreatment of adipocytes with noradrenaline (norepinephrine) caused phosphorylation of AMPK via β-adrenoceptors and not α1- or α2-adrenoceptors. This effect was not β3-adrenoceptor specific, since responses remained intact in adipocytes from β3-adrenoceptor knock-out mice. These effects were also mimicked by forskolin and cAMP analogues. Treatment of cells with adenine 8-β-d-arabinofuranoside, an AMPK inhibitor, partially blocked β-adrenoceptor-mediated increases in glucose uptake. Brown adipocytes are characterised by the production of UCP1, which can uncouple the mitochondria. Using adipocytes from Ucp1+/+ and Ucp1−/− mice, we showed that noradrenaline-mediated phosphorylation of AMPK does not require the presence or activity of UCP1.Conclusions/interpretationThese results suggest a pathway where increases in cAMP mediated by β-adrenoceptors leads to activation of AMPK in brown adipocytes, which contributes in part to β-adrenoceptor-mediated increases in glucose uptake, an effect independent of the presence or function of UCP1.

Altered regulation of the PINK1 locus: a link between type 2 diabetes and neurodegeneration?

Mutations in PINK1 cause the mitochon‐drial‐related neurodegenerative disease Parkinsons. Here we investigate whether obesity, type 2 diabetes, or inactivity alters transcription from the PINK1 locus. We utilized a cDNA‐array and quantitative real‐time PCR for gene expression analysis of muscle from healthy volunteers following physical inactivity, and muscle and adipose tissue from nonobese or obese subjects with normal glucose tolerance or type 2 diabetes. Functional studies of PINK1 were performed utilizing RNAinterference in cell culture models. Following inactivity, the PINK1 locus had an opposing regulation pattern (PINK1 was down‐regu‐lated while natural antisense PINK1 was up‐regulated). In type 2 diabetes skeletal muscle, all transcripts from the PINK1 locus were suppressed and gene expression correlated with diabetes status. RNA interference of PINK1 in human neuronal cell lines impaired basal glucose uptake. In adipose tissue, mitochondrial gene expression correlated with PINK1 expression although remained unaltered following siRNA knockdown of Pink1 in primary cultures of brown preadipocytes. In conclusion, regulation of the PINK1 locus, previously linked to neurodegen‐erative disease, is altered in obesity, type 2 diabetes and inactivity, while the combination of RNAi experiments and clinical data suggests a role for PINK1 in cell energetics rather than in mitochondrial biogenesis.—Scheele C., Nielsen, A. R., Walden, T. B., Sewell, D. A., Fischer, C. P., Brogan, R. J., Petrovic, N., Larsson, O., Tesch, P. A., Wennmalm, K., Hutchinson, D. S., Cannon, B., Wahlestedt C., Pedersen, B. K., Timmons J. A. Altered regulation of the PINK1 locus: a link between Type 2 diabetes and neurodegeneration? FASEB J. 21, 3653–3665 (2007)

Regulation of AMP-activated protein kinase activity by G-protein coupled receptors : Potential utility in treatment of diabetes and heart disease

G-protein coupled receptors (GPCRs) comprise the largest and most diverse family of membrane receptors in the human genome, relaying information from a vast array of external stimuli. GPCRs are targets for approximately 30% of all current therapeutic agents. Recently some GPCRs have been shown to mediate part of their effects through activation of AMP-activated protein kinase (AMPK), a sensor of whole body energy status that plays a pivotal role in whole body energy balance by integrating signals in the periphery and central nervous system. It regulates glucose and lipid metabolism, food intake and body weight, making it an attractive target for the treatment of diseases such as type 2 diabetes and obesity. It mediates the effects of several important adipokines such as leptin and adiponectin and is thought to be responsible for the antidiabetic effects of metformin and thiazolidinediones. A diverse number of GPCRs (including adrenoceptors, cannabinoid receptors, ghrelin receptors, melanocortin receptors) modulate AMPK activity. This review focuses on the regulation of AMPK by GPCRs and signaling intermediates of GPCR signaling such as cyclic AMP and calcium, and how GPCR signaling can modulate AMPK activity by several different mechanisms, and the therapeutic implications of AMPK activation by GPCRs.

Ligand-directed signaling at the beta3-adrenoceptor produced by 3-(2-Ethylphenoxy)-1-[(1,S)-1,2,3,4-tetrahydronapth-1-ylamino]-2S-2-propanol oxalate (SR59230A) relative to receptor agonists.

This study examines signaling pathways activated by the mouse β3-adrenoceptor (AR) expressed in Chinese hamster ovary cells at high (CHOβ3H) or low (CHOβ3L) levels. Functional responses included extracellular acidification rate (ECAR), cAMP accumulation, and p38 mitogen-activated protein kinase (MAPK) or extracellular signal-regulated protein kinase 1/2 (Erk1/2) phosphorylation. (–)-Isoproterenol and the β3-AR agonist (R, R)-5-[2-[[2-(3-chlorophenyl)-2-hydroxyethyl]-amino]-propyl]1,3-benzodioxole-2,2-decarboxylate (CL316243) caused concentration-dependent increases in cAMP accumulation and ECAR in CHOβ3H and CHOβ3L cells. For cAMP accumulation, the β3-AR ligand SR59230A was a partial agonist in CHOβ3H and an antagonist in CHOβ3L cells but for ECAR was an agonist at both expression levels. This suggested that SR59230A, which is normally regarded as an antagonist, can selectively activate pathways leading to ECAR. Examination of the pathways stimulated by (–)-isoproterenol, CL316243, and SR59230A for both ECAR and cAMP accumulation suggested that the cAMP pathway predominates in CHOβ3H cells, whereas p38 MAPK is a major contributor to ECAR in CHOβ3L cells and was the sole contributor to responses to SR59230A. Western blots of p38 MAPK and Erk1/2 phosphorylation confirmed that MAPKs are activated in CHOβ3H and CHOβ3L cells by CL316243 and SR59230A but that SR59230A has much higher efficacy. In addition, p38 MAPK phosphorylation displayed differences in drug potency and efficacy between CHOβ3H and CHOβ3L cells related to inhibition of the response by cAMP. Thus, CL316243 and SR59230A display reversed orders of efficacy for cAMP accumulation compared with Erk1/2 and p38 MAPK phosphorylation, providing a strong indication of ligand-directed signaling.

Glucose uptake in brown fat cells is dependent on mTOR complex 2–promoted GLUT1 translocation

β3-Adrenoceptors promote glucose uptake in brown adipose tissue via both cAMP-mediated increases in GLUT1 transcription and mTORC2-stimulated translocation of newly synthesized GLUT1 to the plasma membrane.

NORADRENALINE RELEASE IN THE LOCUS COERULEUS MODULATES MEMORY FORMATION AND CONSOLIDATION; ROLES FOR α- AND β-ADRENERGIC RECEPTORS

Noradrenaline, essential for the modulation of memory, is released in various parts of the brain from nerve terminals controlled by the locus coeruleus (LoC). Noradrenaline release consequent upon input from higher brain areas also occurs within the LoC itself. We examined the effect of noradrenaline on adrenergic receptors in the LoC on memory processing, using colored bead discrimination learning in the young domestic chick. We have shown previously that the release of noradrenaline in the hippocampus and cortex (mesopallium) is essential for acquisition and consolidation of short-term to intermediate and to long-term memory. Noradrenaline release within the LoC is triggered by the glutamatergic input from the forebrain. Inhibition by LoC injection of NMDA or AMPA receptor antagonists is rescued by injection of β2-and β3-adrenoceptor (AR) agonists in the hippocampus. We show that inhibition of α2A-ARs by BRL44408 in the LoC up to 30 min post-training consolidates weakly-reinforced learning. Conversely activation of α2A-ARs in the LoC at the times of consolidation between short-term and intermediate and long-term memory caused memory loss, which is likely to be due to a decreased release of noradrenaline within these two time windows. The α2A-AR antagonist will block presynaptic inhibitory receptors leading to an increase in extracellular noradrenaline. This interpretation is supported by the actions of noradrenaline uptake blockers that produce the same memory outcome. BRL44408 in the mesopallium also caused memory enhancement. β2-ARs are important in the first time window, whereas α1-, α2C-and β3-ARs are important in the second time window. The results reveal that for successful memory formation noradrenaline release is necessary within the LoC as well as in other brain regions, at the time of consolidation of memory from short-term to intermediate and from intermediate to long-term memory.

Role of β-Adrenoceptors in Memory Consolidation : β3-Adrenoceptors Act on Glucose Uptake and β2-Adrenoceptors on Glycogenolysis

Noradrenaline, acting via β2- and β3-adrenoceptors (AR), enhances memory formation in single trial-discriminated avoidance learning in day-old chicks by mechanisms involving changes in metabolism of glucose and/or glycogen. Earlier studies of memory consolidation in chicks implicated β3- rather than β2-ARs in enhancement of memory consolidation by glucose, but did not elucidate whether stimulation of glucose uptake or of glycolysis was responsible. This study examines the role of glucose transport in memory formation using central injection of the nonselective facilitative glucose transporter (GLUT) inhibitor cytochalasin B, the endothelial/astrocytic GLUT-1 inhibitor phloretin and the Na+/energy-dependent endothelial glucose transporter (SGLT) inhibitor phlorizin. Cytochalasin B inhibited memory when injected into the mesopallium (avian cortex) either close to or between 25 and 45 min after training, whereas phloretin and phlorizin only inhibited memory at 30 min. This suggested that astrocytic/endothelial (GLUT-1) transport is critical at the time of consolidation, whereas a different transporter, probably the neuronal glucose transporter (GLUT-3), is important at the time of training. Inhibition of glucose transport by cytochalasin B, phloretin, or phlorizin also interfered with β3-AR-mediated memory enhancement 20 min posttraining, whereas inhibition of glycogenolysis interfered with β2-AR agonist enhancement of memory. We conclude that in astrocytes (1) activities of both GLUT-1 and SGLT are essential for memory consolidation 30 min posttraining; (2) neuronal GLUT-3 is essential at the time of training; and (3) β2- and β3-ARs consolidate memory by different mechanisms; β3-ARs stimulate central glucose transport, whereas β2-ARs stimulate central glycogenolysis.

Mouse β3a‐ and β3b‐adrenoceptors expressed in Chinese hamster ovary cells display identical pharmacology but utilize distinct signalling pathways

This study characterizes the mouse β3a‐adrenoceptor (AR) and the splice variant of the β3‐AR (β3b‐AR) expressed in Chinese hamster ovary cells (CHO‐K1). Stable clones with high (∼1200), medium (∼500) or low receptor expression (∼100 fmol mg protein−1) were determined by saturation binding with [125I]‐(−)‐cyanopindolol. Competition binding studies showed no significant differences in affinity of β‐AR ligands for either receptor. Several functional responses of each receptor were measured, namely extracellular acidification rate (EAR; cytosensor microphysiometer), cyclic AMP accumulation, and Erk1/2 phosphorylation. The β3‐AR agonists BRL37344, CL316243, GR265162X, L755507, SB251023, the non‐conventional partial β‐AR agonist CGP12177 and the β‐AR agonist (−)‐isoprenaline caused concentration‐dependent increases in EAR in cells expressing either splice variant. CL316243 caused concentration‐dependent increases in cyclic AMP accumulation and Erk1/2 phosphorylation in cells expressing either receptor. PTX treatment increased maximum EAR and cyclic AMP responses to CL316243 in cells expressing the β3b‐AR but not in cells expressing the β3a‐AR at all levels of receptor expression. CL316243 increased Erk1/2 phosphorylation with pEC50 values and maximum responses that were not significantly different in cells expressing either splice variant. Erk1/2 phosphorylation was insensitive to PTX or H89 (PKA inhibitor) but was inhibited by LY294002 (PI3Kγ inhibitor), PP2 (c‐Src inhibitor), genistein (tyrosine kinase inhibitor) and PD98059 (MEK inhibitor). The adenylate cyclase activators forskolin or cholera toxin failed to increase Erk1/2 levels although both treatments markedly increased cyclic AMP accumulation in both β3a‐ or β3b‐AR transfected cells. These results suggest that in CHO‐K1 cells, the β3b‐AR, can couple to both Gs and Gi to stimulate and inhibit cyclic AMP production respectively, while the β3a‐AR, couples solely to Gs to increase cyclic AMP levels. However, the increase in Erk1/2 phosphorylation following receptor activation is not dependent upon coupling of the receptors to Gi or the generation of cyclic AMP.