MCT expression and lactate influx/efflux in tanycytes involved in glia-neuron metabolic interaction

Metabolic interaction via lactate between glial cells and neurons has been proposed as one of the mechanisms involved in hypothalamic glucosensing. We have postulated that hypothalamic glial cells, also known as tanycytes, produce lactate by glycolytic metabolism of glucose. Transfer of lactate to neighboring neurons stimulates ATP synthesis and thus contributes to their activation. Because destruction of third ventricle (III-V) tanycytes is sufficient to alter blood glucose levels and food intake in rats, it is hypothesized that tanycytes are involved in the hypothalamic glucose sensing mechanism. Here, we demonstrate the presence and function of monocarboxylate transporters (MCTs) in tanycytes. Specifically, MCT1 and MCT4 expression as well as their distribution were analyzed in Sprague Dawley rat brain, and we demonstrate that both transporters are expressed in tanycytes. Using primary tanycyte cultures, kinetic analyses and sensitivity to inhibitors were undertaken to confirm that MCT1 and MCT4 were functional for lactate influx. Additionally, physiological concentrations of glucose induced lactate efflux in cultured tanycytes, which was inhibited by classical MCT inhibitors. Because the expression of both MCT1 and MCT4 has been linked to lactate efflux, we propose that tanycytes participate in glucose sensing based on a metabolic interaction with neurons of the arcuate nucleus, which are stimulated by lactate released from MCT1 and MCT4-expressing tanycytes.     

Purinergic signaling in tanycytes and its contribution to nutritional sensing

Tanycytes are hypothalamic radial glial-like cells with an important role in the regulation of neuroendocrine axes and energy homeostasis. These cells have been implicated in glucose, amino acids, and fatty acid sensing in the hypothalamus of rodents, where they are strategically positioned. While their cell bodies contact the cerebrospinal fluid, their extensive processes contact neurons of the arcuate and ventromedial nuclei, protagonists in the regulation of food intake. A growing body of evidence has shown that purinergic signaling plays a relevant role in this homeostatic role of tanycytes, likely regulating the release of gliotransmitters that will modify the activity of satiety-controlling hypothalamic neurons. Connexin hemichannels have proven to be particularly relevant in these mechanisms since they are responsible for the release of ATP from tanycytes in response to nutritional signals. On the other hand, either ionotropic or metabotropic ATP receptors are involved in the generation of intracellular Ca²⁺ waves in response to hypothalamic nutrients, which can spread between glial cells and towards neighboring neurons. This review will summarize recent evidence that supports a nutrient sensor role for tanycytes, highlighting the participation of purinergic signaling in this process.     

MCT2 Expression and Lactate Influx in Anorexigenic and Orexigenic Neurons of the Arcuate Nucleus

Hypothalamic neurons of the arcuate nucleus control food intake, releasing orexigenic and anorexigenic neuropeptides in response to changes in glucose concentration. Several studies have suggested that the glucosensing mechanism is governed by a metabolic interaction between neurons and glial cells via lactate flux through monocarboxylate transporters (MCTs). Hypothalamic glial cells (tanycytes) release lactate through MCT1 and MCT4; however, similar analyses in neuroendocrine neurons have yet to be undertaken. Using primary rat hypothalamic cell cultures and fluorimetric assays, lactate incorporation was detected. Furthermore, the expression and function of MCT2 was demonstrated in the hypothalamic neuronal cell line, GT1-7, using kinetic and inhibition assays. Moreover, MCT2 expression and localization in the Sprague Dawley rat hypothalamus was analyzed using RT-PCR, in situ hybridization and Western blot analyses. Confocal immunohistochemistry analyses revealed MCT2 localization in neuronal but not glial cells. Moreover, MCT2 was localized to ∼90% of orexigenic and ∼60% of anorexigenic neurons as determined by immunolocalization analysis of AgRP and POMC with MCT2-positives neurons. Thus, MCT2 distribution coupled with lactate uptake by hypothalamic neurons suggests that hypothalamic neurons control food intake using lactate to reflect changes in glucose levels.     

Nesfatin-1 influences the excitability of glucosensing neurons in the hypothalamic nuclei and inhibits the food intake

Nesfatin-1 is a recently discovered neuropeptide that has been shown to decrease food intake after lateral, third, or fourth brain ventricle, cisterna magna administration, or PVN injection in ad libitum fed rats. With regards to the understanding of nesfatin-1 brain sites of action, additional microinjection studies will be necessary to define specific nuclei, in addition to the PVN, responsive to nesfatin-1 to get insight into the differential effects on food intake. In the present study, we evaluated nesfatin-1 action to modulate food intake response upon injection into the specific hypothalamic nuclei (PVN, LHA and VMN) in freely fed rats during the dark phase. We extend previous observations by showing that the nesfatin-1 (50 pmol) injected before the onset of the dark period significantly reduced the 1 to 5 h cumulative food intake in rats cannulated into the PVN, LHA, but not in rats cannulated into the VMN. Glucosensing neurons located in the hypothalamus are involved in glucoprivic feeding and homeostatic control of blood glucose. In order to shed light on the mechanisms by which nesfatin-1 exerts its satiety-promoting actions, we examined the effect of nesfatin-1 on the excitability of hypothalamic glucosensing neurons. Nesfatin-1 excited most of the glucose-inhibited (GI) neurons and inhibited most of the glucose-excited (GE) neurons in the PVN. Of 34 GI neurons in the LHA tested, inhibitory effects were seen in 70.6% (24/34) of GI neurons. The main effects were excitatory after intra-VMN administration of nesfatin-1 in GE neurons (27/35, 77.1%). Thus, our data clearly demonstrate that nesfatin-1 may exert at least a part of its physiological actions on the control of food intake as a direct result of its role in modulating the excitability of glucosensing neurons in the PVN, LHA and VMN.     

Hypothalamic ependymal-glial cells express the glucose transporter GLUT2, a protein involved in glucose sensing

The GLUT2 glucose transporter and the K-ATP-sensitive potassium channels have been implicated as an integral part of the glucose-sensing mechanism in the pancreatic islet beta cells. The expression of GLUT2 and K-ATP channels in the hypothalamic region suggest that they are also involved in a sensing mechanism in this area. The hypothalamic glial cells, known as tanycytes alpha and beta, are specialized ependymal cells that bridge the cerebrospinal fluid and the portal blood of the median eminence. We used immunocytochemistry, in situ hybridization and transport analyses to demonstrate the glucose transporters expressed in tanycytes. Confocal microscopy using specific antibodies against GLUT1 and GLUT2 indicated that both transporters are expressed in alpha and beta tanycytes. In addition, primary cultures of mouse hypothalamic tanycytes were found to express both GLUT1 and GLUT2 transporters. Transport studies, including 2-deoxy-glucose and fructose uptake in the presence or absence of inhibitors, indicated that these transporters are functional in cultured tanycytes. Finally, our analyses indicated that tanycytes express the K-ATP channel subunit Kir6.1 in vitro. As the expression of GLUT2 and K-ATP channel is linked to glucose-sensing mechanisms in pancreatic beta cells, we postulate that tanycytes may be responsible, at least in part, for a mechanism that allows the hypothalamus to detect changes in glucose concentrations.     

Purinergic signaling in hypothalamic tanycytes: potential roles in chemosensing

Hypothalamic tanycytes are cells that line the walls of the 3rd ventricle. Their cell bodies contact the cerebrospinal fluid and give rise to an inwardly directed process. The more dorsally located (α1 and α2) tanycytes project to areas of the brain involved in the control of feeding and energy balance (the arcuate nucleus and ventromedial hypothalamic nucleus). Although their functions are poorly understood, they have some similarities to glial cells. Recent evidence shows that they express key molecules involved in purinergic signaling and at least some tanycytes may act as adult multipotent stem cells. Emerging evidence suggests that tanycytes signal through changes in intracellular Ca(2+) and that they can respond with large Ca(2+) signals to ATP and transmitters associated with wakefulness and the drive to feed. They are also glucosensitive and this response is dependent on release of ATP from tanycytes and the activation of P2Y1 receptors. Their ability to release ATP gives potential for their integration into the hypothalamic circuitry controlling energy balance and feeding, but many fundamental questions about their possible functions and roles remain unanswered.     

Elevated expression of glucose transporter-1 in hypothalamic ependymal cells not involved in the formation of the brain-cerebrospinal fluid barrier

Glucose transporters play an essential role in the acquisition of glucose by the brain. Elevated expression of glucose transporter-1 has been detected in endothelial cells of the blood-brain barrier and in choroid plexus cells of the blood-cerebrospinal fluid barrier. On the other hand, there is a paucity of information on the expression of glucose transporters in the ependymal cells that line the walls of the cerebral ventricles. The tanycytes are specialized ependymal cells localized in circumventricular organs such as the median eminence that can be segregated into at least three types, alpha, beta1 and beta2. The beta2 tanycytes form tight junctions and participate in the formation of the cerebrospinal fluid-median eminence barrier. Using immunocytochemistry and in situ hybridization, we analyzed the expression of hexose transporters in rat and mouse hypothalamic tanycytes. In both species, immunocytochemical analysis revealed elevated expression of glucose transporter-1 in alpha and beta1 tanycytes. Intense anti-glucose transporter-1 staining was observed in cell processes located throughout the arcuate nucleus, in the end-feet reaching the lateral sulcus of the infundibular region, and in cell processes contacting the hypothalamic capillaries. On the other hand, there was very low expression of glucose transporter-1 in beta2 tanycytes involved in barrier function. In contrast with the results of the cytochemical analysis, in situ hybridization revealed that tanycytes alpha, beta1, and beta2 express similar levels of glucose transporter-1 mRNA. Further analysis using anti-glial fibrillary acidic protein antibodies to identify areas rich in astrocytes revealed that astrocytes were absent from areas containing alpha and beta1 tanycytes, but were abundant in regions containing the barrier-forming beta2 tanycytes. Overall, our data reveal a lack of correlation between participation in barrier function and expression of glucose transporter-1 in hypothalamic tanycytes. Given the virtual absence of astrocytes in areas rich in alpha and beta1 tanycytes, we speculate whether the tanycytes might have astrocyte-like functions and participate in the metabolic coupling between glia and neurons in the hypothalamic area.     

A golgi study on tanycytes and liquor-contacting cells in the posterior hypothalamus of the newt

Summary Golgi methods were employed to study neurons and ependymal tanycytes in the posterior hypothalamus of the newt. The tanycytes send a few coarse, spiny or barbed processes towards the pia mater. In the periventricular grey, the neurohistological methods show common neurons, ranging from a multipolar to a plumed organization, and abundant liquor-contacting cells. These cells, possibly neurons, give rise to a process that reaches the cerebro-spinal fluid, and terminates in a spindle-shaped swelling, with a thin thread at its tip. In other cells, the intraventricular endings are bulbous or finger-like. The occurrence of: (1) branches of the liquor-contacting process, running parallel to the infundibular surface; (2) infundibular processes which end at the base or between the ependymal cell bodies; and (3) axons coursing in the same position, all indicates that the subependymal layer is a site for complex intercellular relationships. The significance of liquor-contacting cells and tanycytes is discussed, in view of the possibility that they may represent part of a system for hypothalamic regulation in response to changes in the CSF.     

Glucosensing by GnRH neurons: inhibition by androgens and involvement of AMP-activated protein kinase

GnRH neurons integrate steroidal and metabolic cues to regulate fertility centrally. Central glucoprivation reduces LH secretion, which is governed by GnRH release, suggesting GnRH neuron activity is modulated by glucose availability. Here we tested whether GnRH neurons can sense changes in extracellular glucose, and whether glucosensing is altered by the steroids dihydrotestosterone (DHT) and/or estradiol (E). Extracellular recordings were made from GnRH neurons in brain slices from ovariectomized (OVX) mice ± DHT and/or E implants. Firing rate was reduced by a switch from 4.5 to 0.2 mm glucose in cells from OVX, OVX+E, and OVX+DHT+E mice, but not OVX+DHT mice. This suggests that androgens reduce the sensitivity of GnRH neurons to changes in extracellular glucose, but E mitigates this effect. Next we investigated potential mechanisms. In the presence of the ATP-sensitive potassium channel antagonist tolbutamide, glucosensing persisted. In contrast, glucosensing was attenuated in the presence of compound C, an antagonist of AMP-activated protein kinase (AMPK), suggesting a role for AMPK in glucosensing. The AMPK activator N1-(b-D-ribofuranosyl)-5-aminoimidazole-4-carboxamide (AICAR) mimicked the effect of low glucose and was less effective in cells from DHT-treated mice. The effect of DHT to diminish responses to low glucose and AICAR was abolished by blockade of fast synaptic transmission. Both AICAR and low glucose activated a current with a reversal potential near -50 mV, suggesting a nonspecific cation current. These studies indicate that glucosensing is one mechanism by which GnRH neurons sense fuel availability and point to a novel role for AMPK in the central regulation of fertility.     

Neuronal Expression of Glucosylceramide Synthase in Central Nervous System Regulates Body Weight and Energy Homeostasis

Hypothalamic neurons are main regulators of energy homeostasis. Neuronal function essentially depends on plasma membrane-located gangliosides. The present work demonstrates that hypothalamic integration of metabolic signals requires neuronal expression of glucosylceramide synthase (GCS; UDP-glucose:ceramide glucosyltransferase). As a major mechanism of central nervous system (CNS) metabolic control, we demonstrate that GCS-derived gangliosides interacting with leptin receptors (ObR) in the neuronal membrane modulate leptin-stimulated formation of signaling metabolites in hypothalamic neurons. Furthermore, ganglioside-depleted hypothalamic neurons fail to adapt their activity (c-Fos) in response to alterations in peripheral energy signals. Consequently, mice with inducible forebrain neuron-specific deletion of the UDP-glucose:ceramide glucosyltransferase gene (Ugcg) display obesity, hypothermia, and lower sympathetic activity. Recombinant adeno-associated virus (rAAV)-mediated Ugcg delivery to the arcuate nucleus (Arc) significantly ameliorated obesity, specifying gangliosides as seminal components for hypothalamic regulation of body energy homeostasis.     

The role of hypothalamic malonyl-CoA in energy homeostasis

Energy balance is monitored by hypothalamic neurons that respond to peripheral hormonal and afferent neural signals that sense energy status. Recent physiologic, pharmacologic, and genetic evidence has implicated malonyl-CoA, an intermediate in fatty acid synthesis, as a regulatory component of this energy-sensing system. The level of malonyl-CoA in the hypothalamus is dynamically regulated by fasting and feeding, which alter subsequent feeding behavior. Fatty acid synthase (FAS) inhibitors, administered systemically or intracerebroventricularly to lean or obese mice, increase hypothalamic malonyl-CoA leading to the suppression of food intake. Conversely, lowering malonyl-CoA with an acetyl-CoA carboxylase (ACC) inhibitor or by the ectopic expression of malonyl-CoA decarboxylase in the hypothalamus increases food intake and reverses inhibition by FAS inhibitors. Physiologically, the level of hypothalamic malonyl-CoA appears to be determined through phosphorylation/dephosphorylation of ACC by AMP kinase in response to changes in the AMP/ATP ratio, an indicator of energy status. Recent evidence suggests that the brain-specific carnitine:palmitoyl-CoA transferase-1 (CPT1c) may be a regulated target of malonyl-CoA that relays the "malonyl-CoA signal" in hypothalamic neurons that express the orexigenic and anorexigenic neuropeptides that regulate food intake and peripheral energy expenditure. Together these findings support a role for malonyl-CoA as an intermediary in the control of energy homeostasis.     

Prior hypoglycemia enhances glucose responsiveness in some ventromedial hypothalamic glucosensing neurons

Antecedent insulin-induced hypoglycemia (IIH) reduces adrenomedullary responses (AMR) to subsequent bouts of hypoglycemia. The ventromedial hypothalamus [VMH: arcuate (ARC) + ventromedial nuclei] contains glucosensing neurons, which are thought to be mediators of these AMR. Since type 1 diabetes mellitus often begins in childhood, we used juvenile (4- to 5-wk-old) rats to demonstrate that a single bout of IIH (5 U/kg sc) reduced plasma glucose by 24% and peak epinephrine by 59% 1 day later. This dampened AMR was associated with 46% higher mRNA for VMH glucokinase, a key mediator of neuronal glucosensing. Compared with neurons from saline-injected rats, ventromedial nucleus glucose-excited neurons from insulin-injected rats demonstrated a leftward shift in their glucose responsiveness (EC50 = 0.45 and 0.10 mmol/l for saline and insulin, respectively, P = 0.05) and a 31% higher maximal activation by glucose (P = 0.05), although this maximum occurred at a higher glucose concentration (saline, 0.7 vs. insulin, 1.5 mmol/l). Although EC50 values did not differ, ARC glucose-excited neurons had 19% higher maximal activation, which occurred at a lower glucose concentration in insulin- than saline-injected rats (saline, 2.5 vs. insulin, 1.5 mmol/l). In addition, ARC glucose-inhibited neurons from insulin-injected rats were maximally inhibited at a fivefold lower glucose concentration (saline, 2.5 vs. insulin, 0.5 mmol/l), although this inhibition declined at >0.5 mmol/l glucose. These data suggest that the increased VMH glucokinase after IIH may contribute to the increased responsiveness of VMH glucosensing neurons to glucose and the associated blunting of the AMR.     

Hindbrain neurons as an essential hub in the neuroanatomically distributed control of energy balance

This Review highlights the processing and integration performed by hindbrain nuclei, focusing on the inputs received by nucleus tractus solitarius (NTS) neurons. These inputs include vagally mediated gastrointestinal satiation signals, blood-borne energy-related hormonal and nutrient signals, and descending neural signals from the forebrain. We propose that NTS (and hindbrain neurons, more broadly) integrate these multiple energy status signals and issue-output commands controlling the behavioral, autonomic, and endocrine responses that collectively govern energy balance. These hindbrain-mediated controls are neuroanatomically distributed; they involve endemic hindbrain neurons and circuits, hindbrain projections to peripheral circuits, and projections to and from midbrain and forebrain nuclei.     

Role of the diencephalic preoptic region in coordination of thermoregulatory and nutrition-related reactions

In acute experiments on cats we studied convergence of the afferent temperature-related and glycemic signals on the neurons of the preoptic region (RPO1). Local heating and cooling (±7°C) of the skin on the contralateral forelimb pad were used for identification ofRPO neurons as thermosensitive units, while infusion of 0.1–0.4 ml of 5.5% glucose solution in the ipsilaterala. carotis revealed their sensitivity to shifts in the glucose concentration. More than half (57%) of glucosensitiveRPO neurons responded to the changes in peripheral temperature. The pattern of convergence and peculiarities of localization of the convergent neurons were studied. We propose the following hypothesis:RPO neurons receiving convergent thermal and glycemic inputs provide formation of integral controlling signals addressed to the ventromedial and lateral hypothalamic nuclei, which control the thermoregulatory food consumption.     

Thyroid hormones, cytokines, physical training and metabolic control.

During the acute training response, peripheral cellular mechanisms are mainly metabolostatic to achieve energy supply. During prolonged training, glycogen deficiency occurs; this is associated with increased expression of local cytokines, and decreased insulin secretion and beta-adrenergic stimulation and lipolysis in adipose tissue which looses energy. This is indicated by decrease of adipocyte hormone leptin, which has inhibitory effects on excitatory hypothalamic neurons. Leptin, insulin, and cytokines such as interleukin 6 (IL-6) contribute to the metabolic error signal to the hypothalamus which result in decrease of hypothalamic release hormones and sympathoadrenergic stimulation. Thyroid stimulating hormone (TSH) is correlated to the metabolic hormones leptin and insulin, and may be used as indicator of metabolic control. Because the hypothalamus integrates various error signals (metabolic, hormonal, sensory afferents, and central stimuli), the pituitary's releasing hormones represent the functional status of an athlete. Long-term overtraining will lead to downregulation of hypothalamic hormonal and sympathoadrenergic responses, catabolism, and fatigue. These changes contribute to myopathy with predominant expression of slow muscle fiber type and inadequacy in performance. Thyroid hormones are closely involved in the training response and metabolic control.     

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.     

From white to beige: a new hypothalamic pathway

Understanding how brown and beige adipocytes can be differentially controlled and activated by neuronal circuits is a fundamental prerequisite to fully comprehend the metabolic role that fat tissue plays in energy homeostasis. In this issue of EMBO reports, Wang et al 1 identify a new hypothalamic route that drives the exclusive recruitment of beige fat via the selective control of sympathetic nervous system (SNS) outflow to subcutaneous white adipose tissue. Since the data strongly suggest that the APPL2–AMPK signaling axis is crucial for this activation, this finding sheds a new light on the cross talk between peripheral homeostatic signals and neurons that are part of hypothalamic energy homeostasis regulatory pathways in the ventromedial hypothalamus (VHM) proposing a new defending mechanism to cold and obesity.     

The rate-limiting step for glucose transport into the hypothalamus is across the blood–hypothalamus interface

Specialized glucosensing neurons are present in the hypothalamus, some of which neighbor the median eminence, where the blood–brain barrier has been reported leaky. A leaky blood–brain barrier implies high tissue glucose levels and obviates a role for endothelial glucose transporters in the control of hypothalamic glucose concentration, important in understanding the mechanisms of glucose sensing We therefore addressed the question of blood–brain barrier integrity at the hypothalamus for glucose transport by examining the brain tissue-to-plasma glucose ratio in the hypothalamus relative to other brain regions. We also examined glycogenolysis in hypothalamus because its occurrence is unlikely in the potential absence of a hypothalamus–blood interface. Across all regions the concentration of glucose was comparable at a given plasma glucose concentration and was a near linear function of plasma glucose. At steady-state, hypothalamic glucose concentration was similar to the extracellular hypothalamic glucose concentration reported by others. Hypothalamic glycogen fell at a rate of ∼1.5 μmol/g/h and remained present in substantial amounts. We conclude for the hypothalamus, a putative primary site of brain glucose sensing that: the rate-limiting step for glucose transport into brain cells is at the blood–hypothalamus interface, and that glycogenolysis is consistent with a substantial blood -to- intracellular glucose concentration gradient.     

Hypothalamic malonyl-coenzyme A and the control of energy balance

An intermediate in the fatty acid biosynthetic pathway, malonyl-coenzyme A (CoA), has emerged as a major regulator of energy homeostasis not only in peripheral metabolic tissues but also in regions of the central nervous system that control satiety and energy expenditure. Fluctuations in hypothalamic malonyl-CoA lead to changes in food intake and peripheral energy expenditure in a manner consistent with an anorexigenic signaling intermediate. Hypothalamic malonyl-CoA is regulated by nutritional and endocrine cues including glucose and leptin, respectively. That malonyl-CoA is an essential component in the energy homeostatic signaling system of the hypothalamus is supported by convergence of physiological, pharmacological, and genetic evidence. This review will focus on evidence implicating malonyl-CoA as a central player in the control of body weight and adiposity as well as clues to the molecular mechanism by which carbon flux through the fatty acid biosynthetic pathway is linked to the neural control of energy balance.