Independent Effects of γ-Aminobutyric Acid Transaminase (GABAT) on Metabolic and Sleep Homeostasis

Breakdown of the major sleep-promoting neurotransmitter, γ-aminobutyric acid (GABA), in the GABA shunt generates catabolites that may enter the tricarboxylic acid cycle, but it is unknown whether catabolic by-products of the GABA shunt actually support metabolic homeostasis. In Drosophila, the loss of the specific enzyme that degrades GABA, GABA transaminase (GABAT), increases sleep, and we show here that it also affects metabolism such that flies lacking GABAT fail to survive on carbohydrate media. Expression of GABAT in neurons or glia rescues this phenotype, indicating a general metabolic function for this enzyme in the brain. As GABA degradation produces two catabolic products, glutamate and succinic semialdehyde, we sought to determine which was responsible for the metabolic phenotype. Through genetic and pharmacological experiments, we determined that glutamate, rather than succinic semialdehyde, accounts for the metabolic phenotype of gabat mutants. This is supported by biochemical measurements of catabolites in wild-type and mutant animals. Using in vitro labeling assays, we found that inhibition of GABAT affects energetic pathways. Interestingly, we also observed that gaba mutants display a general disruption in bioenergetics as measured by altered levels of tricarboxylic acid cycle intermediates, NAD⁺/NADH, and ATP levels. Finally, we report that the effects of GABAT on sleep do not depend upon glutamate, indicating that GABAT regulates metabolic and sleep homeostasis through independent mechanisms. These data indicate a role of the GABA shunt in the development of metabolic risk and suggest that neurological disorders caused by altered glutamate or GABA may be associated with metabolic disruption.     

Long-Term Increased Carnitine Palmitoyltransferase 1A Expression in Ventromedial Hypotalamus Causes Hyperphagia and Alters the Hypothalamic Lipidomic Profile

Lipid metabolism in the ventromedial hypothalamus (VMH) has emerged as a crucial pathway in the regulation of feeding and energy homeostasis. Carnitine palmitoyltransferase (CPT) 1A is the rate-limiting enzyme in mitochondrial fatty acid β-oxidation and it has been proposed as a crucial mediator of fasting and ghrelin orexigenic signalling. However, the relationship between changes in CPT1A activity and the intracellular downstream effectors in the VMH that contribute to appetite modulation is not fully understood. To this end, we examined the effect of long-term expression of a permanently activated CPT1A isoform by using an adeno-associated viral vector injected into the VMH of rats. Peripherally, this procedure provoked hyperghrelinemia and hyperphagia, which led to overweight, hyperglycemia and insulin resistance. In the mediobasal hypothalamus (MBH), long-term CPT1AM expression in the VMH did not modify acyl-CoA or malonyl-CoA levels. However, it altered the MBH lipidomic profile since ceramides and sphingolipids increased and phospholipids decreased. Furthermore, we detected increased vesicular γ-aminobutyric acid transporter (VGAT) and reduced vesicular glutamate transporter 2 (VGLUT2) expressions, both transporters involved in this orexigenic signal. Taken together, these observations indicate that CPT1A contributes to the regulation of feeding by modulating the expression of neurotransmitter transporters and lipid components that influence the orexigenic pathways in VMH.     

Synthesis and release of GABA in cerebral cortical neurons co-cultured with astrocytes from cerebral cortex or cerebellum

Cerebral cortical neurons were co-cultured for up to 7 days with astrocytes after plating on top of a confluent layer of astrocytes cultured from either cerebral cortex or cerebellum (sandwich co-cultures). Neurons co-cultured with either cortical or cerebellar astrocytes showed a high stimulus coupled release of gamma-aminobutyric acid (GABA), which is the neurotransmitter of these neurons. When the astrocyte selective GABA uptake inhibitor 4,5,6,7-tetrahydroisoxazolo[4,5-c]pyridin-3-ol was added during the release experiments, an increase in the stimulus coupled GABA release was seen, indicating that the astrocytes take up a large fraction of GABA released from the neurons. The activity of the GABA synthesizing enzyme glutamate decarboxylase, which is a specific marker of GABAergic neurons, was markedly increased in sandwich co-cultures of cortical neurons and cerebellar astrocytes compared to neurons cultured in the absence of astrocytes whereas in co-cultures with cortical astrocytes this increase was less pronounced. Pure astrocyte cultures did not show any detectable glutamate decarboxylase activity. The astrocyte specific marker enzyme glutamine synthetase (GS) was present at high activity in a glucocorticoid-inducible form in pure astrocytes as well as in co-cultures regardless of the regional origin of the astrocytes. When neurons were cultured on top of the astrocytes, the specific activity of GS was lower compared to astrocytes cultured alone, a result compatible with the notion that neurons are devoid of this enzyme. The results show that cortical neurons develop and differentiate when seeded on top of both homotypic and heterotypic astrocytes. Moreover, it could be demonstrated that the two cell types in the culture system communicate with each other with regard to GABA homeostasis during transmitter release.     

In situ measurements of enzyme activities in the brain

Summary The present review focusses on enzymes involved in the metabolism of amino acid neurotransmitters and the microphotometric determinations of their activities in various layers of the rat hippocampus. The enzymes are NAD-linked isocitrate dehydrogenase (NAD-ICDH), glutamate dehydrogenase (GDH), and GABA transaminase (GABAT), all of which are localized in mitochondria. GDH seems to be restricted to astrocytes, whereas NAD-ICDH and GABAT are localized in neurons as well as in astrocytes. NAD-ICDH is an important enzyme of the tricarboxylic acid cycle and may deliver -ketoglutarate for the formation of glutamate and GABA, which serve as neurotransmitters in the hippocampus. GDH catalyses the interconversion of -ketoglutarate and glutamate, whereas GABAT is the important GABA-degrading enzyme and requires -ketoglutarate for its activity. While differing in their cellular distribution and activity levels, NAD-ICDH, GDH and GABAT are significantly correlated in their hippocampal distribution. Furthermore, developmental and pharmacohistochemical studies suggest that the distribution and activity of astrocytic GDH is correlated with amino-acidergic neurotransmission in the hippocampus. The data reported give further evidence for a metabolic relationship between neurons and astrocytes in the turnover and metabolism of glutamate and GABA.     

Microphotometric determination of enzymes in brain sections. II. GABA transaminase

The tetrazolium salt procedure of van Gelder (1965) for the demonstration of GABA transaminase (GABAT; the most important GABA degrading enzyme) was adapted for microphotometric measurements of GABAT activities in brain sections using the hippocampus of rats as selected brain region. The final incubation medium consisted of 50 mM GABA, 5 mM alpha-ketoglutarate, 7 mM NAD, 10 mM sodium azide, 6 mM nitroblue tetrazolium chloride, 20 mM malonate and 15% polyvinyl alcohol in 0.05 M Hepes buffer; the final pH was 8.0. There was a linear relationship between GABAT activity and section thickness up to 14 microns and between GABAT activity and reaction time at least up to 20 min (kinetic and end-point measurements). Phenazine methosulfate as an exogenous electron carrier and pyridoxal-5-phosphate as coenzyme of GABAT did not enhance the demonstrable GABAT activities, whereas sodium azide as a blocker of the respiratory chain resulted in an increase of demonstrable enzyme activities. A coreaction of succinate dehydrogenase was excluded by the use of malonate (competitive inhibitor). Using the incubation medium described GABAT activities were demonstrated via the endogenous enzymes succinic semialdehyde dehydrogenase and NADH tetrazolium reductase which were shown to be not rate limiting and seems to be similarly localized as GABAT.     

Glucose inhibits GABA release by pancreatic beta-cells through an increase in GABA shunt activity

GABA is the major inhibitory neurotransmitter in the nervous system. It is also released by the insulin-producing beta-cells, providing them with a potential paracrine regulator. Because glucose was found to inhibit GABA release, we investigated whether extracellular GABA can serve as a marker for glucose-induced mitochondrial activity and thus for the functional state of beta-cells. GABA release by rat and human beta-cells was shown to reflect net GABA production, varying with the functional state of the cells. Net GABA production is the result of GABA formation through glutamate decarboxylase (GAD) and GABA catabolism involving a GABA-transferase (GABA-T)-mediated shunt to the TCA cycle. GABA-T exhibits K(m) values for GABA (1.25 mM) and for alpha-ketoglutarate (alpha-KG; 0.49 mM) that are, respectively, similar to and lower than those in brain. The GABA-T inhibitor gamma-vinyl GABA was used to assess the relative contribution of GABA formation and catabolism to net production and release. The nutrient status of the beta-cells was found to regulate both processes. Glutamine dose-dependently increased GAD-mediated formation of GABA, whereas glucose metabolism shunts part of this GABA to mitochondrial catabolism, involving alpha-KG-induced activation of GABA-T. In absence of extracellular glutamine, glucose also contributed to GABA formation through aminotransferase generation of glutamate from alpha-KG; this stimulatory effect increased GABA release only when GABA-T activity was suppressed. We conclude that GABA release from beta-cells is regulated by glutamine and glucose. Glucose inhibits glutamine-driven GABA formation and release through increasing GABA-T shunt activity by its cellular metabolism. Our data indicate that GABA release by beta-cells can be used to monitor their metabolic responsiveness to glucose irrespective of their insulin-secretory activity.     

Inhibition of hypothalamic carnitine palmitoyltransferase-1 decreases food intake and glucose production

The enzyme carnitine palmitoyltransferase-1 (CPT1) regulates long-chain fatty acid (LCFA) entry into mitochondria, where the LCFAs undergo beta-oxidation. To investigate the mechanism(s) by which central metabolism of lipids can modulate energy balance, we selectively reduced lipid oxidation in the hypothalamus. We decreased the activity of CPT1 by administering to rats a ribozyme-containing plasmid designed specifically to decrease the expression of this enzyme or by infusing pharmacological inhibitors of its activity into the third cerebral ventricle. Either genetic or biochemical inhibition of hypothalamic CPT1 activity was sufficient to substantially diminish food intake and endogenous glucose production. These results indicated that changes in the rate of lipid oxidation in selective hypothalamic neurons signaled nutrient availability to the hypothalamus, which in turn modulated the exogenous and endogenous inputs of nutrients into the circulation.     

Localization and effect of ectopic expression of CPT1c in CNS feeding centers

Hypothalamic neurons monitor peripheral energy status and produce signals to adjust food intake and energy expenditure to maintain homeostasis. However, the molecular mechanisms by which these signals are generated remain unclear. Fluctuations in the level of hypothalamic malonyl-CoA are known to serve as an intermediary in regulating energy homeostasis and it has been proposed that the brain-specific carnitine palmitoyltransferase-1c (CPT1c) serves as a target of malonyl-CoA in the central nervous system (CNS). Here, we report that CPT1c is widely expressed in neurons throughout the CNS including the hypothalamus, hippocampus, cortex, and amygdala. CPT1c is enriched in neural feeding centers of the hypothalamus with mitochondrial localization as an outer integral membrane protein. Ectopic over-expression of CPT1c by stereotactic hypothalamic injection of a CPT1c adenoviral vector is sufficient to protect mice from body weight gain when fed a high-fat diet. These findings show that CPT1c is appropriately localized in regions and cell types to regulate energy homeostasis and that its over-expression in the hypothalamus is sufficient to protect mice from adverse weight gain caused by high-fat intake.     

Microphotometric determination of enzymes in brain sections. II. GABA transaminase

Summary The tetrazolium salt procedure of van Gelder (1965) for the demonstration of GABA transaminase (GABAT; the most important GABA degrading enzyme) was adapted for microphotometric measurements of GABAT activities in brain sections using the hippocampus of rats as selected brain region. The final incubation medium consisted of 50 mM GABA, 5 mM α-ketoglutarate, 7 mM NAD, 10 mM sodium azide, 6 mM nitroblue tetrazolium chloride, 20 mM malonate and 15% polyvinyl alcohol in 0.05 M Hepes buffer; the final pH was 8.0. There was a linear relationship between GABAT activity and section thickness up to 14 μm and between GABAT activity and reaction time at least up to 20 min (kinetic and end-point measurements). Phenazine methosulfate as an exogenous electron carrier and pyridoxal-5-phosphate as coenzyme of GABAT did not enhance the demonstrable GABAT activities, whereas sodium azide as a blocker of the respiratory chain resulted in an increase of demonstrable enzyme activities. A coreaction of succinate dehydrogenase was excluded by the use of malonate (competitive inhibitor). Using the incubation medium described GABAT activities were demonstrated via the endogenous enzymes succinic semialdehyde dehydrogenase and NADH tetrazolium reductase which were shown to be not rate limiting and seems to be similarly localized as GABAT. Supported by the Deutsche Forschungsgemeinschaft (Ku 541/2-2)     

The distribution and mechanism of action of ghrelin in the CNS demonstrates a novel hypothalamic circuit regulating energy homeostasis

The gastrointestinal peptide hormone ghrelin stimulates appetite in rodents and humans via hypothalamic actions. We discovered expression of ghrelin in a previously uncharacterized group of neurons adjacent to the third ventricle between the dorsal, ventral, paraventricular, and arcuate hypothalamic nuclei. These neurons send efferents onto key hypothalamic circuits, including those producing neuropeptide Y (NPY), Agouti-related protein (AGRP), proopiomelanocortin (POMC) products, and corticotropin-releasing hormone (CRH). Within the hypothalamus, ghrelin bound mostly on presynaptic terminals of NPY neurons. Using electrophysiological recordings, we found that ghrelin stimulated the activity of arcuate NPY neurons and mimicked the effect of NPY in the paraventricular nucleus of the hypothalamus (PVH). We propose that at these sites, release of ghrelin may stimulate the release of orexigenic peptides and neurotransmitters, thus representing a novel regulatory circuit controlling energy homeostasis.     

CPT1A-mediated fatty acid oxidation promotes cell proliferation via nucleoside metabolism in nasopharyngeal carcinoma

As the first rate-limiting enzyme in fatty acid oxidation (FAO), CPT1 plays a significant role in metabolic adaptation in cancer pathogenesis. FAO provides an alternative energy supply for cancer cells and is required for cancer cell survival. Given the high proliferation rate of cancer cells, nucleotide synthesis gains prominence in rapidly proliferating cells. In the present study, we found that CPT1A is a determining factor for the abnormal activation of FAO in nasopharyngeal carcinoma (NPC) cells. CPT1A is highly expressed in NPC cells and biopsies. CPT1A dramatically affects the malignant phenotypes in NPC, including proliferation, anchorage-independent growth, and tumor formation ability in nude mice. Moreover, an increased level of CPT1A promotes core metabolic pathways to generate ATP, inducing equivalents and the main precursors for nucleotide biosynthesis. Knockdown of CPT1A markedly lowers the fraction of ¹³C-palmitate-derived carbons into pyrimidine. Periodic activation of CPT1A increases the content of nucleoside metabolic intermediates promoting cell cycle progression in NPC cells. Targeting CPT1A-mediated FAO hinders the cell cycle G1/S transition. Our work verified that CPT1A links FAO to cell cycle progression in NPC cellular proliferation, which supplements additional experimental evidence for developing a therapeutic mechanism based on manipulating lipid metabolism.Subject terms: Cancer metabolism, Cell growth     

Changes in the electroencephalographic and gamma-aminobutyric acid transaminase and succinic semialdehyde dehydrogenase in the allergen induced rat brain

Electroencephalographic activity and gamma-Aminobutyric acid Transaminase together with Succinic semialdehyde dehydrogenase activity changes produced by sensitization with Prosopis juliflora pollen allergen were studied in the cerebral cortex and hypothalamus of the rat brain. Electrical activity of EEG recording begins to appear on 3rd day after sensitization with maximum increase in activity was found on day 9 and decreased after that. A sudden increase in electrical activity was produced in 9th day sensitized rat with 10 min after giving challenging dose intravenously. The measurement of enzymatic activity of GABA-T and SSA-DH showed decrease and increase in 3, 9, 15 and 30 days sensitized rat hypothalamus and cerebral cortex whole homogenate and mitochondrial fractions. A maximum changes in enzymatic activity was found in 9th day sensitized rat with significant alterations after giving sudden stress as challenging dose. These changes in EEG activity and GABA-ergic neurotransmitter in allergenic rats showed the immunoregulatory role of nervous system mediated via GABA shunt.     

Subcellular Location and Neuronal Release of Diazepam Binding Inhibitor

Diazepam binding inhibitor (DBI), a peptide located in CNS neurons, blocks the binding of benzodiazepines and beta-carbolines to the allosteric modulatory sites of gamma-aminobutyric acid (GABAA) receptors. Subcellular fractionation studies of rat brain indicate that DBI is compartmentalized. DBI-like immunoreactivity is highly enriched in synaptosomes obtained by differential centrifugation in isotonic sucrose followed by a Percoll gradient. In synaptosomal lysate, DBI-like immunoreactivity is primarily associated with synaptic vesicles partially purified by differential centrifugation and continuous sucrose gradient. Depolarization induced by high K+ levels (50 mM) or veratridine (50 microM) released DBI stored in neurons of superfused slices of hypothalamus, hippocampus, striatum, and cerebral cortex. The high K+ level-induced release is Ca2+ dependent, and the release induced by veratridine is blocked by 1.7 microM tetrodotoxin. Depolarization released GABA and Met5-enkephalin-Arg6-Phe7 together with DBI. DBI is also released by veratridine depolarization, in a tetrodotoxin-sensitive fashion, from primary cultures of cerebral cortical neurons, but not from cortical astrocytes. Depolarization fails to release DBI from slices of liver and other peripheral organs. These data support the view that DBI may be released as a putative neuromodulatory substance from rat brain neurons.     

Cpt1a silencing in AgRP neurons improves cognitive and physical capacity and promotes healthy aging in male mice

Orexigenic neurons expressing agouti-related protein (AgRP) and neuropeptide Y in the arcuate nucleus (ARC) of the hypothalamus are activated in response to dynamic variations in the metabolic state, including exercise. We previously observed that carnitine palmitoyltransferase 1a (CPT1A), a rate-limiting enzyme of mitochondrial fatty acid oxidation, is a key factor in AgRP neurons, modulating whole-body energy balance and fluid homeostasis. However, the effect of CPT1A in AgRP neurons in aged mice and during exercise has not been explored yet. We have evaluated the physical and cognitive capacity of adult and aged mutant male mice lacking Cpt1a in AgRP neurons (Cpt1a KO). Adult Cpt1a KO male mice exhibited enhanced endurance performance, motor coordination, locomotion, and exploration compared with control mice. No changes were observed in anxiety-related behavior, cognition, and muscle strength. Adult Cpt1a KO mice showed a reduction in gastrocnemius and tibialis anterior muscle mass. The cross-sectional area (CSA) of these muscles were smaller than those of control mice displaying a myofiber remodeling from type II to type I fibers. In aged mice, changes in myofiber remodeling were maintained in Cpt1a KO mice, avoiding loss of physical capacity during aging progression. Additionally, aged Cpt1a KO mice revealed better cognitive skills, reduced inflammation, and oxidative stress in the hypothalamus and hippocampus. In conclusion, CPT1A in AgRP neurons appears to modulate health and protects against aging. Future studies are required to clarify whether CPT1A is a potential antiaging candidate for treating diseases affecting memory and physical activity.     

The Expression of GHS-R in Primary Neurons Is Dependent upon Maturation Stage and Regional Localization

Ghrelin is a hormone with a crucial role in the regulation of appetite, regulation of inflammation, glucose metabolism and cell proliferation. In the brain ghrelin neurons are located in the cortex (sensorimotor area, cingular gyrus), and the fibres of ghrelin neurons in hypothalamus project directly to the dorsal vagal complex (DVC). Ghrelin binds the growth hormone secretagogue receptor (GHS-R) a G-protein-coupled receptor with a widespread tissue distribution, indeed these receptors are localized both in nonnervous, organs/tissues (i.e. adipose tissue, myocardium, adrenals, gonads, lung, liver, arteries, stomach, pancreas, thyroid, and kidney) as well as in central nervous system (CNS) and higher levels of expression in the pituitary gland and the hypothalamus and lower levels of expression in other organs, including brain. A GHS-R specific monoclonal antibody has been developed and characterized and through it we demonstrate that GHS-R is expressed in primary neurons and that its expression is dependent upon their developmental stage and shows differences according to the brain region involved, with a more pronounced expression in hippocampal rather than cortical neurons. A characterization of GHS-R within the central nervous system is of extreme importance in order to gain insights on its role in the modulation of neurodegenerative events such as Alzheimer’s disease.     

Ghrelin-containing neuron in cerebral cortex and hypothalamus linked with the DVC of brainstem in rat

Ghrelin is a newly discovered brain-gut peptide and an endogenous ligand for growth hormone secretagogues receptor (GHS-R). Ghrelin and GHS-R present extensively in central and peripheral tissues such as stomach, brain and other organs of rodent and human, which suggest it has multiple biological effects. It has been reported that ghrelin has significant role in the regulation of energy homeostasis, food intake and appetite. The organization of central circuitry appears to play an important role in integrating orexigenic effects of ghrelin, but the detail is not fully clear. In this study, we examined the expression of ghrelin, ghrelin mRNA and GHS-R mRNA in cerebrum and brainstem by RT-PCR and immunofluorescence histochemistry, and analyzed the connection among the cerebral cortex, hypothalamus, dorsal vagal complex (DVC). The results showed that the positive staining of ghrelin was found on the pyramidal neuron of layer V in the sensorimotor area of cerebral cortex, cingulate gyrus, as well as in the neuron of lateral hypothalamus (LH), PVN and ARC. The expression of ghrelin mRNA and GHS-R mRNA were also found in the sensorimotor cortex and hypothalamus by method of RT-PCR. The GHS-R mRNA was also found in the DVC of medulla oblongata. Other finding is that the FG/ghrelin dual labeled neurons were found in LH of hypothalamus (not in cortex). The ghrelin-containing neuron in the LH projects its axon to the DVC with the method of retrograde tracing. In conclusion, the ghrelin neurons are located not only in hypothalamus (LH, PVN, ARC), but also in the cortex (sensorimotor area, cingular gyrus), and the fibers of ghrelin neurons in hypothalamus projected directly to the DVC. It suggests that ghrelin plays its role from hypothalamus to brainstem as a neurotransmitter or neuromodulator to regulate function of vagal nuclei in brainstem.     

The orexigenic effect of ghrelin is mediated through central activation of the endogenous cannabinoid system

Introduction

Ghrelin and cannabinoids stimulate appetite, this effect possibly being mediated by the activation of hypothalamic AMP-activated protein kinase (AMPK), a key enzyme in appetite and metabolism regulation. The cannabinoid receptor type 1 (CB1) antagonist rimonabant can block the orexigenic effect of ghrelin. In this study, we have elucidated the mechanism of the putative ghrelin-cannabinoid interaction.

Methods

The effects of ghrelin and CB1 antagonist rimonabant in wild-type mice, and the effect of ghrelin in CB1-knockout animals, were studied on food intake, hypothalamic AMPK activity and endogenous cannabinoid content. In patch-clamp electrophysiology experiments the effect of ghrelin was assessed on the synaptic inputs in parvocellular neurons of the hypothalamic paraventricular nucleus, with or without the pre-administration of a CB1 antagonist or of cannabinoid synthesis inhibitors.

Results and Conclusions

Ghrelin did not induce an orexigenic effect in CB1-knockout mice. Correspondingly, both the genetic lack of CB1 and the pharmacological blockade of CB1 inhibited the effect of ghrelin on AMPK activity. Ghrelin increased the endocannabinoid content of the hypothalamus in wild-type mice and this effect was abolished by rimonabant pre-treatment, while no effect was observed in CB1-KO animals. Electrophysiology studies showed that ghrelin can inhibit the excitatory inputs on the parvocellular neurons of the paraventricular nucleus, and that this effect is abolished by administration of a CB1 antagonist or an inhibitor of the DAG lipase, the enzyme responsible for 2-AG synthesis. The effect is also lost in the presence of BAPTA, an intracellular calcium chelator, which inhibits endocannabinoid synthesis in the recorded parvocellular neuron and therefore blocks the retrograde signaling exerted by endocannabinoids. In summary, an intact cannabinoid signaling pathway is necessary for the stimulatory effects of ghrelin on AMPK activity and food intake, and for the inhibitory effect of ghrelin on paraventricular neurons.     

Hypothalamic fatty acid metabolism mediates the orexigenic action of ghrelin

Current evidence suggests that hypothalamic fatty acid metabolism may play a role in regulating food intake; however, confirmation that it is a physiologically relevant regulatory system of feeding is still incomplete. Here, we use pharmacological and genetic approaches to demonstrate that the physiological orexigenic response to ghrelin involves specific inhibition of fatty acid biosynthesis induced by AMP-activated protein kinase (AMPK) resulting in decreased hypothalamic levels of malonyl-CoA and increased carnitine palmitoyltransferase 1 (CPT1) activity. In addition, we also demonstrate that fasting downregulates fatty acid synthase (FAS) in a region-specific manner and that this effect is mediated by an AMPK and ghrelin-dependent mechanisms. Thus, decreasing AMPK activity in the ventromedial nucleus of the hypothalamus (VMH) is sufficient to inhibit ghrelin's effects on FAS expression and feeding. Overall, our results indicate that modulation of hypothalamic fatty acid metabolism specifically in the VMH in response to ghrelin is a physiological mechanism that controls feeding.     

The Effect of γ-Aminobutyric Acid on Substrate-Level Phosphorylation in Brain Mitochondria

Abstract: A consequence of the metabolism of γ-aminobutyric acid (GABA) via the "GABA shunt" should be a decreased rate of substrate-level phos- phorylation of GDP to GTP. ³²P₁ labeling of nucleotides was, therefore, studied in uncoupled brain mitochondria with α-ketoglutarate or a-ketoglutarate + GABA as substrates. The addition of an equimolar amount of GABA resulted in an approximately 50% reduction of the final specific activity of all mitochondrial nucleotides. This effect was completely reversed by aminooxyacetic acid. GABA did not affect the time course of nucleotide labeling. Although delineation of the mechanism involved requires further study, these preliminary results suggest an important modulatory role of GABA in the intermediary metabolism of brain mitochondria.     

Modulation of GABA-augmented norepinephrine release in female rat brain slices by opioids and adenosine

GABA_A receptor activation augments electrically-stimulated release of norepinephrine (NE) from rat brain slices. Because this effect is not observed in synaptoneurosomes, GABA probably acts on inhibitory interneurons to disinhibit NE release. To determine whether opioids or adenosine influence GABA-augmented NE release, hypothalamic and cortical slices from female rats were superfused with GABA or vehicle in the presence and absence of 10 M morphine or 100 M adenosine. GABA augments [³H]NE release in the cortex and hypothalamus. Morphine alone has no effect on [³H]NE release, but attenuates GABA augmentation of [³H]NE release in both brain regions. Adenosine alone modestly inhibits [³H]NE release in the cortex, but not in the hypothalamus. Adenosine inhibits GABA-augmented [³H]NE release in both brain regions. The general protein kinase inhibitor H-7, augments [³H]NE release in both brain regions and may have additive effects with GABA in cortical slices. These results implicate opioid and adenosine interneurons and possibly protein kinases in regulating GABAergic influences on NE transmission.