Hyperglycemia impairs EAAT2 glutamate transporter trafficking and glutamate clearance in islets of Langerhans: implications for type 2 diabetes pathogenesis and treatment.

Pancreatic endocrine cells employ a sophisticated system of paracrine and autocrine signals to synchronize their activities, including glutamate, which controls hormone release and β-cell viability by acting on glutamate receptors expressed by endocrine cells. We here investigate whether alteration of the excitatory amino acid transporter 2 (EAAT2), the major glutamate clearance system in the islet, may occur in type 2 diabetes mellitus and contribute to β-cell dysfunction. Increased EAAT2 intracellular localization was evident in islets of Langerhans from T2DM subjects as compared with healthy control subjects, despite similar expression levels.

Cholesterol Redistribution in Pancreatic β-Cells: A Flexible Path to Regulate Insulin Secretion.

Pancreatic β-cells, by secreting insulin, play a key role in the control of glucose homeostasis, and their dysfunction is the basis of diabetes development. The metabolic milieu created by high blood glucose and lipids is known to play a role in this process. In the last decades, cholesterol has attracted significant attention, not only because it critically controls β-cell function but also because it is the target of lipid-lowering therapies proposed for preventing the cardiovascular complications in diabetes.

Pancreatic PCSK9 controls the organization of the β-cell secretory pathway via LDLR-cholesterol axis.

Background: Cholesterol is central to pancreatic β-cell physiology and alterations of its homeostasis contribute to β-cell dysfunction and diabetes. Proper intracellular cholesterol levels are maintained by different mechanisms including uptake via the low-density lipoprotein receptor (LDLR). In the liver, the proprotein convertase subtilisin/kexin type 9 (PCSK9) routes the LDLR to lysosomes for degradation, thus limiting its recycling to the membrane.

Iron Metabolism in Pancreatic Beta-Cell Function and Dysfunction.

Iron is an essential element involved in a variety of physiological functions. In the pancreatic beta-cells, being part of Fe-S cluster proteins, it is necessary for the correct insulin synthesis and processing. In the mitochondria, as a component of the respiratory chain, it allows the production of ATP and reactive oxygen species (ROS) that trigger beta-cell depolarization and potentiate the calcium-dependent insulin release.

The Lepidopteran KAAT1 and CAATCH1: Orthologs to Understand Structure-Function Relationships in Mammalian SLC6 Transporters.

To the SLC6 family belong 20 human transporters that utilize the sodium electrochemical gradient to move biogenic amines, osmolytes, amino acids and related compounds into cells. They are classified into two functional groups, the Neurotransmitter transporters (NTT) and Nutrient amino acid transporters (NAT). Here we summarize how since their first cloning in 1998, the insect (Lepidopteran) Orthologs of the SLC6 family transporters have represented very important tools for investigating functional-structural relationships, mechanism of transport, ion and pH dependence and substate interaction of the mammalian (and human) counterparts.

Characterization of Transport Activity of SLC11 Transporters in Oocytes by Fluorophore Quenching.

Membrane proteins are involved in different physiological functions and are the target of pharmaceutical and abuse drugs. oocytes provide a powerful heterologous expression system for functional studies of these proteins. Typical experiments investigate transport using electrophysiology and radiolabeled uptake.

Verbascoside Protects Pancreatic β-Cells against ER-Stress.

Substantial epidemiological evidence indicates that a diet rich in polyphenols protects against developing type 2 diabetes. The phenylethanoid glycoside verbascoside/acteoside, a widespread polyphenolic plant compound, has several biological properties including strong antioxidant, anti-inflammatory and neuroprotective activities. The aim of this research was to test the possible effects of verbascoside on pancreatic β-cells, a target never tested before.

Neurotransmitters and Neuropeptides: New Players in the Control of Islet of Langerhans' Cell Mass and Function.

Islets of Langerhans control whole body glucose homeostasis, as they respond, releasing hormones, to changes in nutrient concentrations in the blood stream. The regulation of hormone secretion has been the focus of attention for a long time because it is related to many metabolic disorders, including diabetes mellitus. Endocrine cells of the islet use a sophisticate system of endocrine, paracrine and autocrine signals to synchronize their activities.

Dictyostelium Nramp1, which is structurally and functionally similar to mammalian DMT1 transporter, mediates phagosomal iron efflux.

The Nramp (Slc11) protein family is widespread in bacteria and eukaryotes, and mediates transport of divalent metals across cellular membranes. The social amoeba Dictyostelium discoideum has two Nramp proteins. Nramp1, like its mammalian ortholog (SLC11A1), is recruited to phagosomal and macropinosomal membranes, and confers resistance to pathogenic bacteria.

Imaging of Xenopus laevis oocyte plasma membrane in physiological-like conditions by atomic force microscopy.

Xenopus laevis oocytes are an interesting model for the study of many developmental mechanisms because of their dimensions and the ease with which they can be manipulated. In addition, they are widely employed systems for the expression and functional study of heterologous proteins, which can be expressed with high efficiency on their plasma membrane. Here we applied atomic force microscopy (AFM) to the study of the plasma membrane of X.

GABA reverse transport by the neuronal cotransporter GAT1: influence of internal chloride depletion.

The role of intracellular ions on the reverse GABA transport by the neuronal transporter GAT1 was studied using voltage-clamp and [(3)H]GABA efflux determinations in Xenopus oocytes transfected with heterologous mRNA. Reverse transport was induced by intracellular GABA injections and measured in terms of the net outward current generated by the transporter. Changes in various intracellular ionic conditions affected the reverse current: higher concentrations of Na(+) enhanced the ratio of outward over inward transport current, while a considerable decrease of the outward current and a parallel reduction of the transporter-mediated GABA efflux were observed after treatments causing a diminution of the intracellular Cl(-) concentration.

Passive water permeability of some wild type and mutagenized amino acid cotransporters of the SLC6/NSS family expressed in Xenopus laevis oocytes.

In this paper passive water movement across the cell membrane mediated by wild type and mutagenized cotransporters was investigated. We evaluated water movement and, in parallel, amino acid uptake induced by some members of the SLC6/NSS family belonging to different kingdoms, namely the rat GABA transporter GAT1, the insect amino acid transporters KAAT1 and CAATCH1 and the bacterial leucine transporter LeuT, whose structure was recently solved. We also tested whether mutated proteins in which the solute translocation mechanism is altered or even abolished were able to induce water movement across cell membrane.

The SLC6/NSS family members KAAT1 and CAATCH1 have weak chloride dependence.

KAAT1 and CAATCH1 are amino acid transporters cloned from the intestine of the lepidoptera Manduca sexta. They are members of the SLC6/NSS family, which groups membrane proteins that use Na(+), K(+), and Cl⁻ gradients for the coupled transport of amines and amino acids. The report of the atomic-resolution x-ray crystal structure of the eubacterium Aquifex aeolicus leucine transporter (AaLeuT) has contributed significantly to understanding of the structure-function relationship in NSS proteins.

Structural and functional basis of amino acid specificity in the invertebrate cotransporter KAAT1.

The substrate specificity of KAAT1, a Na+- and K+-dependent neutral amino acid cotransporter cloned from the larva of the invertebrate Manduca sexta and belonging to the SLC6A gene family has been investigated using electrophysiological and radiotracer methods. The specificity of KAAT1 was compared to that of CAATCH1, a strictly related transporter with different amino acid selectivity. Competition experiments between different substrates indicate that both transporters bind leucine more strongly than threonine and proline, the difference between KAAT1 and CAATCH1 residing in the incapacity of the latter to complete the transport cycle in presence of leucine.

Structural domains involved in substrate selectivity in two neutral amino acid transporters.

The ability of the two highly homologous Na(+)/Cl(-)-dependent neutral amino acid transporters KAAT1 and CAATCH1, cloned from the midgut epithelium of the larva Manduca sexta, to transport different amino acids depends on the cotransported ion, on pH, and on the membrane voltage. Different organic substrates give rise to transport-associated currents with their own characteristics, which are notably distinct between the two proteins. Differences in amplitude, kinetics, and voltage dependence of the transport-associated currents have been observed, as well as different substrate selectivity patterns measured by radioactive amino acid uptake assays.

Glutamate 59 is critical for transport function of the amino acid cotransporter KAAT1.

KAAT1 is a neutral amino acid transporter activated by K+ or by Na+ (9). The protein shows significant homology with members of the Na+/Cl--dependent neurotransmitter transporter super family. E59G KAAT1, expressed in Xenopus oocytes, exhibited a reduced leucine uptake [20-30% of wild-type (WT)], and kinetic analysis indicated that the loss of activity was due to reduction of Vmax and apparent affinity for substrates.

Inhibition of the lepidopteran amino acid co-transporter KAAT1 by phenylglyoxal: role of arginine 76.

Phenylglyoxal (PGO), an arginine-modifying reagent, is an irreversible inhibitor of KAAT1-mediated leucine transport, expressed in Xenopus oocytes. The PGO effect was dose-dependent and 5 mm PGO determined a V(max) reduction to 24% of the control, consistent with the covalent binding to transporter arginine residues not located in the leucine binding site. The use of labelled [(14)C]PGO confirmed that the inhibitor binds KAAT1.