Ca2+ permeation through C-terminal cleaved, but not full-length human Pannexin1 hemichannels, mediates cell death.

Pannexin1 hemichannels (Panx1 HCs) are found in the membrane of most mammalian cells and communicate the intracellular and extracellular spaces, enabling the passive transfer of ions and small molecules. They are involved in physiological and pathophysiological conditions. During apoptosis, the C-terminal tail of Panx1 is proteolytically cleaved, but the permeability features of hemichannels and their role in cell death remain elusive. To address these topics, HeLa cells transfected with full-length human Panx1 (fl-hPanx1) or C-terminal truncated hPanx1 (Δ371hPanx1) were exposed to alkaline extracellular saline solution, increasing the activity of Panx1 HCs. The Δ371hPanx1 HC was permeable to DAPI and Etd+, but not to propidium iodide, whereas fl-hPanx1 HC was only permeable to DAPI. Furthermore, the cytoplasmic Ca2+ signal increased only in Δ371hPanx1 cells, which was supported by bioinformatics approaches. The influx of Ca2+ through Δ371hPanx1 HCs was necessary to promote cell death up to about 95% of cells, whereas the exposure to alkaline saline solution without Ca2+ failed to induce cell death, and the Ca2+ ionophore A23187 promoted more than 80% cell death even in fl-hPanx1 transfectants. Moreover, cell death was prevented with carbenoxolone or 10Panx1 in Δ371hPanx1 cells, whereas it was undetectable in HeLa Panx1-/- cells. Pretreatment with Ferrostatin-1 and necrostatin-1 did not prevent cell death, suggesting that ferroptosis or necroptosis was not involved. In comparison, zVAD-FMK, a pancaspase inhibitor, reduced death by ~60%, suggesting the involvement of apoptosis. Therefore, alkaline pH increases the activity of Δ371hPanx1HCs, leading to a critical intracellular free-Ca2+ overload that promotes cell death.

Unnexin is a protein subunit of a large-pore channel expressed by unicellular organisms.

Cells of vertebrate and invertebrate organisms express proteins specialized in membrane channel-based cell-cell communication that are absent in unicellular organisms. We recently described the prediction of some members of the large-pore channel family in kinetoplastids, consisting of proteins called unnexins, which share several structural features with innexin and pannexin proteins. Here, we demonstrated that the unnexin1 protein (Unx1) is delivered to the cell membrane, displaying a topology consisting of four transmembrane domains with C and N termini on the cytoplasmic side and form large-pore channels that are permeable to small molecules. Low extracellular Ca2+/Mg2+ levels or extracellular alkalinization, but not mechanical stretching, increases channel activity. The Unx1 channel mediates the influx of Ca2+ and does not form intercellular dye coupling between HeLa Unx1 transfected cells. Unx1 channel function was further evidenced by its ability to mediate ionic currents when expressed in Xenopus oocytes. Downregulation of Unx1 mRNA with morpholine contains Trypanosoma cruzi invasion. Phylogenetic analysis revealed the presence of Unx1 homologs in other protozoan parasites, suggesting a conserved function for these channel parasites in other protists. Our data demonstrate that Unx1 forms large-pore membrane channels, which may serve as a diffusional pathway for ions and small molecules that are likely to be metabolic substrates or waste products, and signaling autocrine and paracrine molecules that could be involved in cell invasion. As morpholinos-induced downregulation of Unx1 reduces the infectivity of trypomastigotes, the Unx1 channels might be an attractive target for developing trypanocide drugs.

The connexin hemichannel inhibitor D4 produces rapid antidepressant-like effects in mice.

Depression is a common mood disorder characterized by a range of clinical symptoms, including prolonged low mood and diminished interest. Although many clinical and animal studies have provided significant insights into the pathophysiology of depression, current treatment strategies are not sufficient to manage this disorder. It has been suggested that connexin (Cx)-based hemichannels are candidates for depression intervention by modifying the state of neuroinflammation. In this study, we investigated the antidepressant-like effect of a recently discovered selective Cx hemichannel inhibitor, a small organic molecule called D4. We first showed that D4 reduced hemichannel activity following systemic inflammation after LPS injections. Next, we found that D4 treatment prevented LPS-induced inflammatory response and depressive-like behaviors. These behavioral effects were accompanied by reduced astrocytic activation and hemichannel activity in depressive-like mice induced by repeated low-dose LPS challenges. D4 treatment also reverses depressive-like symptoms in mice subjected to chronic restraint stress (CRS). To test whether D4 broadly affected neural activity, we measured c-Fos expression in depression-related brain regions and found a reduction in c-Fos+ cells in different brain regions. D4 significantly normalized CRS-induced hypoactivation in several brain regions, including the hippocampus, entorhinal cortex, and lateral septum. Together, these results indicate that blocking Cx hemichannels using D4 can normalize neuronal activity and reduce depressive-like symptoms in mice by reducing neuroinflammation. Our work provides evidence of the antidepressant-like effect of D4 and supports glial Cx hemichannels as potential therapeutic targets for depression.

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.

Glial hypothalamic inhibition of GLUT2 expression alters satiety, impacting eating behavior.

Glucose is a key modulator of feeding behavior. By acting in peripheral tissues and in the central nervous system, it directly controls the secretion of hormones and neuropeptides and modulates the activity of the autonomic nervous system. GLUT2 is required for several glucoregulatory responses in the brain, including feeding behavior, and is localized in the hypothalamus and brainstem, which are the main centers that control this behavior. In the hypothalamus, GLUT2 has been detected in glial cells, known as tanycytes, which line the basal walls of the third ventricle (3V). This study aimed to clarify the role of GLUT2 expression in tanycytes in feeding behavior using 3V injections of an adenovirus encoding a shRNA against GLUT2 and the reporter EGFP (Ad-shGLUT2). Efficient in vivo GLUT2 knockdown in rat hypothalamic tissue was demonstrated by qPCR and Western blot analyses. Specificity of cell transduction in the hypothalamus and brainstem was evaluated by EGFP-fluorescence and immunohistochemistry, which showed EGFP expression specifically in ependymal cells, including tanycytes. The altered mRNA levels of both orexigenic and anorexigenic neuropeptides suggested a loss of response to increased glucose in the 3V. Feeding behavior analysis in the fasting-feeding transition revealed that GLUT2-knockdown rats had increased food intake and body weight, suggesting an inhibitory effect on satiety. Taken together, suppression of GLUT2 expression in tanycytes disrupted the hypothalamic glucosensing mechanism, which altered the feeding behavior.

Skeletal Muscle Atrophy Induced by Diabetes Is Mediated by Non-Selective Channels and Prevented by Boldine.

Individuals with diabetes mellitus present a skeletal muscle myopathy characterized by atrophy. However, the mechanism underlying this muscular alteration remains elusive, which makes it difficult to design a rational treatment that could avoid the negative consequences in muscles due to diabetes. In the present work, the atrophy of skeletal myofibers from streptozotocin-induced diabetic rats was prevented with boldine, suggesting that non-selective channels inhibited by this alkaloid are involved in this process, as has previously shown for other muscular pathologies. Accordingly, we found a relevant increase in sarcolemma permeability of skeletal myofibers of diabetic animals in vivo and in vitro due to de novo expression of functional connexin hemichannels (Cx HCs) containing connexins (Cxs) 39, 43, and 45. These cells also expressed P2X7 receptors, and their inhibition in vitro drastically reduced sarcolemma permeability, suggesting their participation in the activation of Cx HCs. Notably, sarcolemma permeability of skeletal myofibers was prevented by boldine treatment that blocks Cx43 and Cx45 HCs, and now we demonstrated that it also blocks P2X7 receptors. In addition, the skeletal muscle alterations described above were not observed in diabetic mice with myofibers deficient in Cx43/Cx45 expression. Moreover, murine myofibers cultured for 24 h in high glucose presented a drastic increase in sarcolemma permeability and levels of NLRP3, a molecular member of the inflammasome, a response that was also prevented by boldine, suggesting that, in addition to the systemic inflammatory response found in diabetes, high glucose can promote the expression of functional Cx HCs and activation of the inflammasome in skeletal myofibers. Therefore, Cx43 and Cx45 HCs play a critical role in myofiber degeneration, and boldine could be considered a potential therapeutic agent to treat muscular complications due to diabetes.

A Short-Term Sucrose Diet Impacts Cell Proliferation of Neural Precursors in the Adult Hypothalamus.

Radial glia-like cells in the hypothalamus and dorsal vagal complex are neural precursors (NPs) located near subventricular organs: median eminence and area postrema, respectively. Their strategic position can detect blood-borne nutrients, hormones, and mitogenic signals. Hypothalamic NPs increase their proliferation with a mechanism that involves hemichannel (HC) activity. NPs can originate new neurons in response to a short-term high-fat diet as a compensatory mechanism. The effects of high carbohydrate Western diets on adult neurogenesis are unknown. Although sugars are usually consumed as sucrose, more free fructose is now incorporated into food items. Here, we studied the proliferation of both types of NPs in Sprague Dawley rats exposed to a short-term high sucrose diet (HSD) and a control diet. In tanycyte cultures, we evaluated the effects of glucose and fructose and a mix of both hexoses on HC activity. In rats fed an HSD, we observed an increase in the proliferative state of both precursors. Glucose, either in the presence or absence of fructose, but not fructose alone, induced in vitro HC activity. These results should broaden the understanding of the nutrient monitoring capacity of NPs in reacting to changes in feeding behavior, specifically to high sugar western diets.

GKRP-dependent modulation of feeding behavior by tanycyte-released monocarboxylates.

Objectives: Glucokinase Regulatory Protein (GKRP) is the only known endogenous modulator of glucokinase (GK) localization and activity to date, and both proteins are localized in tanycytes, radial glia-like cells involved in metabolic and endocrine functions in the hypothalamus. However, the role of tanycytic GKRP and its impact on the regulation of feeding behavior has not been investigated. Here, we hypothesize that GKRP regulates feeding behavior by modulating tanycyte-neuron metabolic communication in the arcuate nucleus. Methods: We used primary cultures of tanycytes to evaluate the production of lactate and ß-hydroxybutyrate (ßHB). Similarly, we examined the electrophysiological responses to these metabolites in pro-opiomelanocortin (POMC) neurons in hypothalamic slices. To evaluate the role of GKRP in feeding behavior, we generated tanycyte-selective GKRP-overexpressing and GKRP-knock down mice (GKRPt-OE and GKRPt-KD respectively) using adenovirus-mediated transduction. Results: We demonstrated that lactate release induced by glucose uptake is favored in GKRP-KD tanycytes. Conversely, tanycytes overexpressing GKRP showed an increase in ßHB efflux induced by low glucose concentration. In line with these findings, the excitability of POMC neurons was enhanced by lactate and decreased in the presence of ßHB. In GKRPt-OE rats, we found an increase in post-fasting food avidity, whereas GKRPt-KD caused a significant decrease in feeding and body weight, which is reverted when MCT1 is silenced. Conclusion: Our study highlights the role of tanycytic GKRP in metabolic regulation and positions this regulator of GK as a therapeutic target for boosting satiety in patients with obesity problems.

The ATP synthase inhibition induces an AMPK-dependent glycolytic switch of mesenchymal stem cells that enhances their immunotherapeutic potential.

Objectives: Mesenchymal Stem/Stromal Cells (MSC) are promising therapeutic tools for inflammatory diseases due to their potent immunoregulatory capacities. Their suppressive activity mainly depends on inflammatory cues that have been recently associated with changes in MSC bioenergetic status towards a glycolytic metabolism. However, the molecular mechanisms behind this metabolic reprogramming and its impact on MSC therapeutic properties have not been investigated. Methods: Human and murine-derived MSC were metabolically reprogramed using pro-inflammatory cytokines, an inhibitor of ATP synthase (oligomycin), or 2-deoxy-D-glucose (2DG). The immunosuppressive activity of these cells was tested in vitro using co-culture experiments with pro-inflammatory T cells and in vivo with the Delayed-Type Hypersensitivity (DTH) and the Graph versus Host Disease (GVHD) murine models. Results: We found that the oligomycin-mediated pro-glycolytic switch of MSC significantly enhanced their immunosuppressive properties in vitro. Conversely, glycolysis inhibition using 2DG significantly reduced MSC immunoregulatory effects. Moreover, in vivo, MSC glycolytic reprogramming significantly increased their therapeutic benefit in the DTH and GVHD mouse models. Finally, we demonstrated that the MSC glycolytic switch effect partly depends on the activation of the AMPK signaling pathway. Conclusion: Altogether, our findings show that AMPK-dependent glycolytic reprogramming of MSC using an ATP synthase inhibitor contributes to their immunosuppressive and therapeutic functions, and suggest that pro-glycolytic drugs might be used to improve MSC-based therapy.

Inhibition of Hypothalamic MCT4 and MCT1-MCT4 Expressions Affects Food Intake and Alters Orexigenic and Anorexigenic Neuropeptide Expressions.

Feeding behavior regulation is a complex process, which depends on the central integration of different signals, such as glucose, leptin, and ghrelin. Recent studies have shown that glial cells known as tanycytes that border the basal third ventricle (3V) detect glucose and then use glucose-derived signaling to inform energy status to arcuate nucleus (ARC) neurons to regulate feeding behavior. Monocarboxylate transporters (MCT) 1 and MCT4 are localized in the cellular processes of tanycytes, which could facilitate monocarboxylate release to orexigenic and anorexigenic neurons. We hypothesize that MCT1 and MCT4 inhibitions could alter the metabolic communication between tanycytes and ARC neurons, affecting feeding behavior. We have previously shown that MCT1 knockdown rats eat more and exhibit altered satiety parameters. Here, we generate MCT4 knockdown rats and MCT1-MCT4 double knockdown rats using adenovirus-mediated transduction of a shRNA into the 3V. Feeding behavior was evaluated in MCT4 and double knockdown animals, and neuropeptide expression in response to intracerebroventricular glucose administration was measured. MCT4 inhibition produced a decrease in food intake, contrary to double knockdown. MCT4 inhibition was accompanied by a decrease in eating rate and mean meal size and an increase in mean meal duration, parameters that are not changed in the double knockdown animals with exception of eating rate. Finally, we observed a loss in glucose regulation of orexigenic neuropeptides and abnormal expression of anorexigenic neuropeptides in response to fasting when these transporters are inhibited. Taken together, these results indicate that MCT1 and MCT4 expressions in tanycytes play a role in feeding behavior regulation.

When a Little Bit More Makes the Difference: Expression Levels of GKRP Determines the Subcellular Localization of GK in Tanycytes.

Glucose homeostasis is performed by specialized cells types that detect and respond to changes in systemic glucose concentration. Hepatocytes, ß-cells and hypothalamic tanycytes are part of the glucosensor cell types, which express several proteins involved in the glucose sensing mechanism such as GLUT2, Glucokinase (GK) and Glucokinase regulatory protein (GKRP). GK catalyzes the phosphorylation of glucose to glucose-6-phosphate (G-6P), and its activity and subcellular localization are regulated by GKRP. In liver, when glucose concentration is low, GKRP binds to GK holding it in the nucleus, while the rise in glucose concentration induces a rapid export of GK from the nucleus to the cytoplasm. In contrast, hypothalamic tanycytes display inverse compartmentalization dynamic in response to glucose: a rise in the glucose concentration drives nuclear compartmentalization of GK. The underlying mechanism responsible for differential GK subcellular localization in tanycytes has not been described yet. However, it has been suggested that relative expression between GK and GKRP might play a role. To study the effects of GKRP expression levels in the subcellular localization of GK, we used insulinoma 832/13 cells and hypothalamic tanycytes to overexpress the tanycytic sequences of Gckr. By immunocytochemistry and Western blot analysis, we observed that overexpression of GKRP, independently of the cellular context, turns GK localization to a liver-like fashion, as GK is mainly localized in the nucleus in response to low glucose. Evaluating the expression levels of GKRP in relation to GK through RT-qPCR, suggest that excess of GKRP might influence the pattern of GK subcellular localization. In this sense, we propose that the low expression of GKRP (in relation to GK) observed in tanycytes is responsible, at least in part, for the compartmentalization pattern observed in this cell type. Since GKRP behaves as a GK inhibitor, the regulation of GKRP expression levels or activity in tanycytes could be used as a therapeutic target to regulate the glucosensing activity of these cells and consequently to regulate feeding behavior.

Author Correction: Adenovirus-mediated suppression of hypothalamic glucokinase affects feeding behavior.

An amendment to this paper has been published and can be accessed via a link at the top of the paper.

Connexin-43 Gap Junctions Are Responsible for the Hypothalamic Tanycyte-Coupled Network.

Tanycytes are hypothalamic radial glia-like cells that form the basal wall of the third ventricle (3V) where they sense glucose and modulate neighboring neuronal activity to control feeding behavior. This role requires the coupling of hypothalamic cells since transient decreased hypothalamic Cx43 expression inhibits the increase of brain glucose-induced insulin secretion. Tanycytes have been postulated as possible hypothalamic neuronal precursors due to their privileged position in the hypothalamus that allows them to detect mitogenic signals and because they share the markers and characteristics of neuronal precursors located in other neurogenic niches, including the formation of coupled networks through connexins. Using wild-type (WT), Cx30-/- and Cx30-/-, Cx43fl/fl:glial fibrillary acidic protein (GFAP)-Cre (double knockout, dKO) mouse lines, we demonstrated that tanycytes are highly coupled to each other and also give rise to a panglial network specifically through Cx43. Using the human GFAP (hGFAP)-enhanced green fluorescent protein (EGFP) transgenic mouse line, we provided evidence that the main parenchymal-coupled cells were astrocytes. In addition, electrophysiological parameters, such as membrane resistance, were altered when Cx43 was genetically absent or pharmacologically inhibited. Finally, in the dKO mouse line, we detected a significant decrease in the number of hypothalamic proliferative parenchymal cells. Our results demonstrate the importance of Cx43 in tanycyte homotypic and panglial coupling and show that Cx43 function influences the proliferative potential of hypothalamic cells.

Adenovirus-mediated suppression of hypothalamic glucokinase affects feeding behavior.

Glucokinase (GK), the hexokinase involved in glucosensing in pancreatic ß-cells, is also expressed in arcuate nucleus (AN) neurons and hypothalamic tanycytes, the cells that surround the basal third ventricle (3V). Several lines of evidence suggest that tanycytes may be involved in the regulation of energy homeostasis. Tanycytes have extended cell processes that contact the feeding-regulating neurons in the AN, particularly, agouti-related protein (AgRP), neuropeptide Y (NPY), cocaine- and amphetamine-regulated transcript (CART) and proopiomelanocortin (POMC) neurons. In this study, we developed an adenovirus expressing GK shRNA to inhibit GK expression in vivo. When injected into the 3V of rats, this adenovirus preferentially transduced tanycytes. qRT-PCR and Western blot assays confirmed GK mRNA and protein levels were lower in GK knockdown animals compared to the controls. In response to an intracerebroventricular glucose injection, the mRNA levels of anorexigenic POMC and CART and orexigenic AgRP and NPY neuropeptides were altered in GK knockdown animals. Similarly, food intake, meal duration, frequency of eating events and the cumulative eating time were increased, whereas the intervals between meals were decreased in GK knockdown rats, suggesting a decrease in satiety. Thus, GK expression in the ventricular cells appears to play an important role in feeding behavior.

Inhibition of hypothalamic MCT1 expression increases food intake and alters orexigenic and anorexigenic neuropeptide expression.

Hypothalamic glucosensing, which involves the detection of glucose concentration changes by brain cells and subsequent release of orexigenic or anorexigenic neuropeptides, is a crucial process that regulates feeding behavior. Arcuate nucleus (AN) neurons are classically thought to be responsible for hypothalamic glucosensing through a direct sensing mechanism; however, recent data has shown a metabolic interaction between tanycytes and AN neurons through lactate that may also be contributing to this process. Monocarboxylate transporter 1 (MCT1) is the main isoform expressed by tanycytes, which could facilitate lactate release to hypothalamic AN neurons. We hypothesize that MCT1 inhibition could alter the metabolic coupling between tanycytes and AN neurons, altering feeding behavior. To test this, we inhibited MCT1 expression using adenovirus-mediated transfection of a shRNA into the third ventricle, transducing ependymal wall cells and tanycytes. Neuropeptide expression and feeding behavior were measured in MCT1-inhibited animals after intracerebroventricular glucose administration following a fasting period. Results showed a loss in glucose regulation of orexigenic neuropeptides and an abnormal expression of anorexigenic neuropeptides in response to fasting. This was accompanied by an increase in food intake and in body weight gain. Taken together, these results indicate that MCT1 expression in tanycytes plays a role in feeding behavior regulation.

Mutagenic and kinetic support for an allosteric site in arginase from the extreme thermophile Bacillus caldovelox, which allows activation by arginine.

To substantiate the functionality of a crystallographically evidenced allosteric site in Bacillus caldovelox arginase (Bewley et al., 1999), we have examined the kinetic consequences of the single mutations of Asp199 and Glu256, which interact with l-arginine in this site. The introduced mutations (Asp199 → Asn, Asp199 → Ala, Glu256 → Gln and Glu256 → Ala) had no effect on the hexameric structure of the enzyme (mol. wt. 195 ± 10 kDa). However, in contrast with the Michaelis-Menten kinetics exhibited by the wild-type species, the D199A, D199N, E256A and E256Q variants exhibited positive cooperativity with respect to l-arginine. The Glu278 → Ala mutation, which compromise interactions at the trimer-trimer interface, yielded trimeric species (mol. wt. 100 ± 5 kDa) exhibiting hyperbolic kinetics that changed to sigmoidal by the additional Glu256 → Ala mutation. In addition to demonstrating the total functionality of the trimer, our results also suggest that B. caldovelox is kinetically cooperative and that the commonly detected hyperbolic behavior results from binding of l-arginine as a typical allosteric activator.

Dynamic localization of glucokinase and its regulatory protein in hypothalamic tanycytes.

Glucokinase (GK), the hexokinase involved in glucose sensing in pancreatic ß cells, is also expressed in hypothalamic tanycytes, which cover the ventricular walls of the basal hypothalamus and are implicated in an indirect control of neuronal activity by glucose. Previously, we demonstrated that GK was preferentially localized in tanycyte nuclei in euglycemic rats, which has been reported in hepatocytes and is suggestive of the presence of the GK regulatory protein, GKRP. In the present study, GK intracellular localization in hypothalamic and hepatic tissues of the same rats under several glycemic conditions was compared using confocal microscopy and Western blot analysis. In the hypothalamus, increased GK nuclear localization was observed in hyperglycemic conditions; however, it was primarily localized in the cytoplasm in hepatic tissue under the same conditions. Both GK and GKRP were next cloned from primary cultures of tanycytes. Expression of GK by Escherichia coli revealed a functional cooperative protein with a S0.5 of 10 mM. GKRP, expressed in Saccharomyces cerevisiae, inhibited GK activity in vitro with a Ki 0.2 µM. We also demonstrated increased nuclear reactivity of both GK and GKRP in response to high glucose concentrations in tanycyte cultures. These data were confirmed using Western blot analysis of nuclear extracts. Results indicate that GK undergoes short-term regulation by nuclear compartmentalization. Thus, in tanycytes, GK can act as a molecular switch to arrest cellular responses to increased glucose.