New Insights into the Determinants of Specificity in Human Type I Arginase: Generation of a Mutant That Is Only Active with Agmatine as Substrate.

Arginase catalyzes the hydrolysis of L-arginine into L-ornithine and urea. This enzyme has several analogies with agmatinase, which catalyzes the hydrolysis of agmatine into putrescine and urea. However, this contrasts with the highlighted specificity that each one presents for their respective substrate. A comparison of available crystal structures for arginases reveals an important difference in the extension of two loops located in the entrance of the active site. The first, denominated loop A (I129-L140) contains the residues that interact with the alpha carboxyl group or arginine of arginase, and the loop B (D181-P184) contains the residues that interact with the alpha amino group of arginine. In this work, to determine the importance of these loops in the specificity of arginase, single, double, and triple arginase mutants in these loops were constructed, as well as chimeras between type I human arginase and E. coli agmatinase. In previous studies, the substitution of N130D in arginase (in loop A) generated a species capable of hydrolyzing arginine and agmatine. Now, the specificity of arginase is completely altered, generating a chimeric species that is only active with agmatine as a substrate, by substituting I129T, N130Y, and T131A together with the elimination of residues P132, L133, and T134. In addition, Quantum Mechanic/Molecular Mechanic (QM/MM) calculations were carried out to study the accommodation of the substrates in in the active site of this chimera. With these results it is concluded that this loop is decisive to discriminate the type of substrate susceptible to be hydrolyzed by arginase. Evidence was also obtained to define the loop B as a structural determinant for substrate affinity. Concretely, the double mutation D181T and V182E generate an enzyme with an essentially unaltered kcat value, but with a significantly increased Km value for arginine and a significant decrease in affinity for its product ornithine.

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.

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.

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.

Molecular Characteristics, Regulation, and Function of Monocarboxylate Transporters.

Lactate transporters play an important role in the glutamate recycling. Here their kinetics and tissue distribution with emphasis on the brain are addressed. Recent evidence shows their participation in important brain functions that involve intercellular communication, such as hypothalamic glucose sensing. Furthermore, we describe the regulation of their expression and some animal models that have allowed clarification of their functions.

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.

Dynamic expression of the sodium-vitamin C co-transporters, SVCT1 and SVCT2, during perinatal kidney development.

Isoform 1 of the sodium-vitamin C co-transporter (SVCT1) is expressed in the apical membrane of proximal tubule epithelial cells in adult human and mouse kidneys. This study is aimed at analyzing the expression and function of SVCTs during kidney development. RT-PCR and immunohistochemical analyses revealed that SVCT1 expression is increased progressively during postnatal kidney development. However, SVCT1 transcripts were barely detected, if not absent, in the embryonic kidney. Instead, the high-affinity transporter, isoform 2 (SVCT2), was strongly expressed in the developing kidney from E15; its expression decreased at postnatal stages. Immunohistochemical analyses showed a dynamic distribution of SVCT2 in epithelial cells during kidney development. In renal cortex tubular epithelial cells, intracellular distribution of SVCT2 was observed at E19 with distribution in the basolateral membrane at P1. In contrast, SVCT2 was localized to the apical and basolateral membranes between E17 and E19 in medullary kidney tubular cells but was distributed intracellularly at P1. In agreement with these findings, functional expression of SVCT2, but not SVCT1 was detected in human embryonic kidney-derived (HEK293) cells. In addition, kinetic analysis suggested that an ascorbate-dependent mechanism accounts for targeted SVCT2 expression in the developing kidney during medullary epithelial cell differentiation. However, during cortical tubular differentiation, SVCT1 was induced and localized to the apical membrane of tubular epithelial cells. SVCT2 showed a basolateral polarization only for the first days of postnatal life. These studies suggest that the uptake of vitamin C mediated by different SVCTs plays differential roles during the ontogeny of kidney tubular epithelial cells.