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REVIEW ARTICLE

The potential role of glutamate in the current diabetes epidemic

Alberto M. Davalli

Carla Perego

Franco B. Folli

Received: 23 August 2011 / Accepted: 19 December 2011 / Published online: 5 January 2012 Springer-Verlag 2012

Abstract In the present article, we propose the perspec-tive that abnormal glutamate homeostasis might contributeto diabetes pathogenesis. Previous reports and our recentdata indicate that chronically high extracellular glutamatelevels exert direct and indirect effects that might participatein the progressive loss of b -cells occurring in both T1D andT2D. In addition, abnormal glutamate homeostasis mayimpact all the three accelerators of the acceleratorhypothesis and could partially explain the rising fre-quency of T1D and T2D.

Keywords L-Glutamic acid Glutamate toxicity Monosodium glutamate Obesity Diabetes

Pancreatic b -cells

Introduction

The incidence of type 1 diabetes (T1D) has been steadilyincreasing worldwide since the middle of the twentiethcentury [ 1, 2], and the increase is highest in very youngchildren [ 2, 3]. A similar scenario relates to type 2 diabetes(T2D) that was once considered a disease of older adults,

while it is now rampant also in children [ 410]. Although itis generally recognized that autoimmune b -cell destructionaccounts for T1D while T2D results from absolute or rel-ative adiposity-related insulin resistance in individuals withlimited b -cell compensatory ability, these disorders havesome overlapping features. In 50% of the cases, T1D isdiagnosed in adulthood, the onset is slow, and many sub- jects do not develop acidosis or require insulin for severalyears [ 11 ]. On the other hand, T2D occurs also in children[2, 3, 12] and sometimes with keto-acidosis [ 13]. More-over, a considerable subgroup of adult and pediatric T2Dsubjects present evidence of humoral and cellular b -cellautoimmunity [ 1418] that is more common than previ-ously thought and that may contribute signicantly to theprogressive decline in b-cell function observed in T2Dpatients. In addition, obesity and insulin resistance are nowrecognized risk factors also for T1D [ 1921], and it is alsoemerging that islet inammation can be present in T2D[22, 23]. Based on this evidence, clinical trials with drugsthat target interleukin-1 b are under investigation in boththe diseases [ 22, 2427].

The accelerator hypothesis postulates that T1D andT2D are indeed the same disease, distinguishable only bythe rate of pancreatic b -cell loss and the presence of theaccelerators responsible for this process [ 28]. Accordingto this hypothesis, the rst accelerator is an intrinsic pre-disposition of the b -cell to undergo apoptosis (a sort of b -cell fragility) that is necessary for diabetes to develop butinsufcient per se to cause it; the second accelerator isinsulin resistance resulting from weight gain and physicalinactivity, which further increases b-cell work and the rateof b -cell apoptosis; and nally, a minor and geneticallypredisposed subset of patients with both intrinsic b -cellfragility and insulin resistance would develop b -cell auto-immunity, which would be the third accelerator. Weight

A. M. Davalli ( & )Diabetes and Endocrinology Unit, Department of InternalMedicine, San Raffaele Scientic Institute, 20132 Milan, Italye-mail: davalli.alberto@hsr.it

A. M. Davalli F. B. FolliDiabetes Division, Department of Medicine, University of TexasHealth Science Center, San Antonio, TX 78229, USA

C. PeregoDepartment of Molecular Science Applied to Biosystems,Universita degli Studi di Milano, 20134 Milan, Italy

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gain and insulin resistance would be thus the missing linksbetween T1D and T2D. The hypothesis that T1D and T2Dare the same disorder of insulin resistance set against dif-ferent genetic backgrounds not only implies commoncausative factors, but also provides a useful speculativelink among the three factors: insulin resistance, b -cell loss,and b -cell autoimmunity. How these factors interact wouldinvolve a wide range of hereditary and environmentalcauses. A changing environment, infant and maternal dietsin particular, is the most likely explanation for the alarmingincrease in diabetes incidence [ 28, 29].

In the present article, we propose that abnormal gluta-mate homeostasis, to which excessive glutamate con-sumption with food may also contribute, could causeelevated extracellular glutamate concentrations that mayparticipate in b -cell death, possibly in combination withincreased FFA and glucose concentrations. Previousreports and our recent data indicate that chronically highextracellular glutamate levels exert direct and indirecteffects that may contribute to the progressive loss of b -cellsoccurring in both T1D and T2D. Abnormal glutamatehomeostasis may impact all the three accelerators of theaccelerator hypothesis and could partially explain therising frequency of T1D and T2D.

Glutamate concentration in biological uids

Glutamate ( L-glutamic acid) is an amino acid present infoods either in free form or in the form of peptides andproteins. Animal protein may contain up to 22% and plantsprotein as much as 40% glutamate by weight. In spite of itsabundance in food, glutamate concentration in blood isamong the lowest. This is mainly because glutamate isextensively oxidized by the small intestine to meet the highenergy demand of the epithelium that is in rapid renewal[30]. In healthy volunteers, nearly all of the entericallydelivered glutamate is removed by the splanchnic bed/liveron the rst pass [ 31]. As a result, glutamate concentrationin the peripheral blood is only about 50 l mol/L, while theconcentration of glutamine, the most abundant amino acidin blood, is about 0.7 mmol/L. Circulating glutamine entersinto the cells where it is enzymatically converted intoglutamate by the action of glutaminase. Glutamate is in factthe most important intracellular amino acid reaching con-centrations up to 20 mmol/L. Intracellular glutamate canbe oxidized to provide energy and is central to numeroustransamination and deamination reactions and to the syn-thesis of proteins. In addition, intracellular glutamateincreases the antioxidant defense by the generation of glutathione and participates in the gluconeogenesis in thekidney and in the urea synthesis in the liver. In GABAergicneurons and pancreatic b-cells, intracellular glutamate may

be also transformed into c-amino butyric acid (GABA) bythe action of the enzyme glutamic acid decarboxylase(GAD), the main autoantigen in T1D and stiff-mansyndrome [ 3234]. Moreover, the discovery that gain-of-function mutation in the gene encoding glutamatedehydrogenases (GDH), the enzyme that converts a -keto-glutarate to glutamate, causes a hyperinsulinemic syndrome[35] supports previous data, indicating that intracellularglutamate acts in b -cells as a second messenger that poten-tiates glucose-induced insulin secretion [ 36].

The plasma glutamate concentration ranges from 50 to100 l mol/L, while in the whole brain, it is about1012 mmol/L even though only 0.52 l mol/L in theextracellular uids. This is because glutamate is the majorexcitatory amino acid neurotransmitter in the mammaliancentral nervous system (CNS) and its concentration in theextracellular uid determines the extent of glutamatereceptor stimulation. There are three types of glutamatereceptors: (1) the ionotropic N-methyl-D-aspartate (NMDA)that has the unique feature of being doubly-gated, requiringmembrane depolarization and ligand binding for activation[37], (2) the ionotropic a -amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA) receptor that mediatesfast synaptic transmission, and (3) the metabotropic glu-tamate receptors (mGluRs) that are G protein coupled andmediate slow synaptic transmission. Ionotropic (iGluRs)and metabotropic (mGluRs) glutamate receptors are foundthroughout the brain but are also expressed in peripheraland extraneural tissues [ 37, 38]. In the CNS, it is of criticalimportance that extracellular glutamate concentration iskept low to maintain a high signal-to-noise (background)ratio in synaptic transmission. Further, excessive activationof both iGluRs and mGluRs is harmful as glutamate istoxic in high concentrations mainly through an excessivecalcium inux and the production of nitric oxide [ 37].Recently, it has also been reported that activation of NADPH oxidase 2 by the stimulation of iGluRs andmGluRs contributes to in vivo glutamate neurotoxicitythrough the production of reactive oxygen species andcalpain activation [ 39].

In the CNS, extracellular glutamate is maintained lowby the action of low- and high-afnity glutamate trans-porters present in neurons and glial cells and by the actionof the bloodbrain barrier (BBB) made of capillary endo-thelial cells that surround the entire CNS. Tight junctionsconnect endothelial cells and separate the BBB into lumi-nal and basolateral domains. Facilitative carriers exist onlyin luminal membranes, while Na ? -dependent glutamatetransporters (excitatory amino acid transporters, EAATs)are exclusively located in abluminal membranes that allowglutamate move from the extracellular uid to the endo-thelial cell where glutamate is free to diffuse into blood onfacilitative carriers [ 40]. This organization prevents net

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glutamate entry to the brain while promotes the removal of glutamate and the maintenance of low glutamate concen-trations in the extracellular uid. Therefore, BBB isimpermeable to glutamate, even at high concentrations,with the notable exception of few small areas that havefenestrated capillaries (circumventricular organs).

iGluRs and mGluRs are expressed also in non-neuronaltissues such as pancreatic islets, hearth, adrenal glands, andlymphocytes [ 38], thus implying that circulating glutamateis not solely a substrate for cell metabolism but it is also animportant signal molecule for the entire body. For instance,pancreatic islets are composed of four types of endocrinecells, that is, insulin-secreting b-cells, glucagon-secretinga -cells, somatostatin-secreting d-cells, and pancreaticpolypeptide-secreting PP-cells, and all of them expressfunctional glutamate receptors [ 4148]. In fact, glutamateinuences the secretion of both insulin and glucagon fromislet cells, isolated islets, or perfused pancreas [ 4148].Glutamate transporters are also widely expressed inperipheral tissues [ 49]. Most cells and tissues have theability to take up glutamate and in particular broblasts,erythrocytes, macrophages, platelets, muscle, prostate,liver, taste buds, mammalian oocytes as well as the heart,intestine, kidney, pancreas, placenta, bone and the mam-mary gland [ 49, 50]. A number of different glutamatetransporters have been identied in the placenta [ 51] wherean intriguing aspect is that glutamate goes in the wrongdirection. Placenta supplies the fetus with most of theamino acids (including glutamine), but removes glutamatefrom the fetal circulation [ 52]. Because fetal BBB is per-meable [ 53, 54], the placental glutamate uptake is impor-tant to avoid dangerous increases in plasma glutamate inthe fetus. It has been hypothesized that the thrifty pheno-type, and the subsequent development of the metabolicsyndrome, may be the consequence of fetal hypergluta-matemia [ 55]. According to this hypothesis, in the presenceof permeable fetal BBB, maternal glutamate would reachneurotoxic levels in the fetus CNS.

Glutamate is the major free amino acid in the humanmilk, representing 2152% of the total amount of thesecomponents [ 56]; it increases during lactation [ 57], and itsconcentration is about 12-fold higher than that in infantformulas [ 58]. Given the low levels of glutamate in thesystemic circulation, the mammary gland acts to concen-trate glutamate in the milk. In Mexican rural lactatingwomen, glutamate was 40-fold higher in milk than inplasma [ 59]. The analysis of milk composition of womenon a typical rural Mexican diet, as compared with thatsecreted by American women consuming a standard dietfor wealthy countries, showed signicantly higher freeglutamate levels in the latter group [ 60]. High free gluta-mate levels in the milk are thought to protect the enteralmucosa and act as neurotransmitters and source of nitrogen

in newborns. However, the great ability of the mammarygland to concentrate glutamate and the impact of the dieton the glutamate content of the maternal milk raise thequestion as to whether high glutamatecontaining diets inlactating women may cause toxic increases in glutamateconcentrations in breast-fed neonates [ 61].

Glutamate toxicity

Low glutamate concentration in the extracellular uids isnecessary for optimal brain function and is maintained byneurons, astrocytes, and the BBB that move glutamateagainst the existing electrochemical gradient [ 40]. In thebrain, extracellular glutamate must be rapidly removedfrom the synaptic cleft to control synaptic events and toprevent the sustained activation of iGluRs, which has apotent neurotoxic effect (so-called excitotoxicity) [ 62]. Inthe CNS, the clearance of extracellular glutamate is carriedout by the glutamate transporters located on the plasmamembrane of astroglial and neuronal cells. Five Na ? -dependent high-afnity excitatory amino acid transporters(EAAT15) have been described [ 49], but EAAT2, alsoknown as glial glutamate transporter of type 1 (GLT1),exhibits the highest level of expression in the CNS [ 63].Mice decient in GLT1 show increased susceptibility toacute cortical injuries [ 64], and inhibition of GLT1 activityincreases the glutamate concentration to toxic levels [ 63].

A classic example of glutamate toxicity is representedby the rodents, which were injected intraperitoneously withmonosodium glutamate at the neonatal stage (the sodiumsalt of glutamic acid, MSG), that are an established animalmodel of obesity, metabolic syndrome, and T2D [ 6570].The large doses of glutamate administered as MSG deter-mine, in the presence of an incomplete BBB, the lesion of the arcuate and ventromedial nuclei that are responsible forthe sense of satiety. Thus, MSG-induced neuronal damageresults in voracity and subsequent excessive weight gain,insulin resistance, and diabetes. However, orally adminis-tered glutamate can hardly induce similar effects. Long-term animal feeding studies have shown that MSG does notincrease food intake or induce obesity [ 71, 72]. Neverthe-less, MSG maintains its toxicity when administered orallyduring pregnancy up to the end of the weaning period inrats [73]. Others have shown that high doses of MSG givenby oral gavage in rats increase plasma and extracellularglutamate in the hippocampus of about fourfold, without,however, causing obvious neurotoxic effects [ 74]. Thus, itis still controversial whether toxic levels of glutamatecould be reached into the CNS of fetuses whose mothersare fed with glutamate-enriched diets. It should bereminded, however, that the medial arcuate nucleus, whichis the area ascribed to feeding control and sensitive to

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glutamate-induced toxicity, belongs to those regions of theCNS that, as perfused by fenestrated capillaries, take upmolecules from the circulation [ 75].

A second example of glutamate toxicity is provided bydiabetic retinopathy. Early in its pathogenesis, a decreasein the ability of Mu ller cells to remove glutamate from theextracellular space disrupts glutamate homeostasis in thediabetic retina [ 76]. As in the CNS, glutamate is toxic toretinal neurons and exacerbates oxidative stress [ 77]. Per-sistent glutamate signaling through the NMDA receptorcontributes to Mu ller cell death, and chronic taurineadministration by increasing the expression of the gluta-mate transporter EAAT-1 improves diabetic retinopathy byreducing glutamate levels in the retina [ 78]. Thereby,taurine supplementation ameliorates diabetic retinopathythrough an anti-excitotoxic mechanism [ 78]. However, ithas been recently reported that, under diabetic conditions,taurine can regulate Muller cells glutamate uptake anddegradation also via its antioxidant effect [ 79].

A third example of glutamate toxicity relates to theintestine. Gastrointestinal tissues are innervated by acomplex and extensive peripheral nervous system knownas the enteric nervous system (ENS) that is composed byenteric neurons (ENs) and enteric glial cells (EGCs). Thelatter show morphologic and functional similarities to CNSastrocytes and form an extensive network of cells in theintestinal mucosa located in close proximity to intestinalepithelial cells and submucosal blood vessels. Glutama-tergic neurons and glutamate-mediated neurotransmissionhave been described in the ENS [ 80]. Glutamate is indeedan excitatory neurotransmitter for the ENS where excessiveexposure to glutamate produces ENs neurotoxicity [ 81].Prolonged stimulation of enteric ganglia by glutamatecauses necrosis and apoptosis in ENs. The occurrence of excitotoxicity in the ENS suggests that overactivation of enteric glutamate receptors may contribute to the intestinaldamage produced by anoxia, ischemia, and excitotoxins(i.e., MSG) present in food [ 81]. EGCs as well expressglutamate receptors [ 82] and, therefore, may be also vul-nerable to excitotoxicity. That EGCs are functional targetsof the glutamatergic neurotransmitter system is furthersupported by the evidence that they express the metabo-tropic glutamate receptor mGlutR5, the expression of which changes in states of inammation [ 83]. In the EGCs,mGlut5R stimulation increases c-Fos and pERK1/2 acti-vation, which could be a pro-apoptotic or a pro-survivalsignal depending on the context [ 83, 84]. The majorphysiologic roles of the EGCs are to provide trophic andcytoprotective functions toward ENs [ 85], maintain theintegrity of the gut mucosa, and regulate its permeabilityand turnover [ 85, 86]. Therefore, glutamate-induced EGCsdysfunction may have profound effects on intestine phys-iology and especially on enteric permeability. Remarkably,

increased permeability has been reported in T1D in humans[8791] and may precede by several years the developmentof diabetes [ 9193].

Glutamate in food

Glutamate is an important contributor to food avors. Thetaste quality associated with glutamate, known as um-ami, is recognized as a unique quality by the gustatorysystem [ 9496]. The umami taste receptor is present intaste buds and responds specically to compounds pos-sessing umami quality [ 97]. The umami receptor has beenlater characterized as a heteromer of the taste-specicT1R1 and T1R3 G protein-coupled receptors (T1R1/3) [ 98,99]. Human T1R1/3 responds to L-glutamate, and theresponse is enhanced by 5 0-ribonucleotides [ 98]. T1R1/3transduction mechanisms include the activation of phos-pholipase C, inositol trisphosphate-mediated release of Ca 2 ? from intracellular stores, and Ca 2 ? -dependent acti-vation of the cation channel TRPM5 [ 100 ]. This series of events lead to membrane depolarization, action potentials,and the release of ATP that acts as transmitter to activategustatory afferents [ 100 ]. Interestingly, the umami receptorhas been recently found also in the b -cell line MIN6 whereits stimulation induces an increase in insulin secretion[101 ]. The ability of umami to elicit characteristic hedonicresponses in human neonates [ 102 ] and across cultures[103 ] is suggestive of a basic taste. Since early 1900s,MSG has been used as a avor enhancer and there is ampleevidence that adding MSG to foods increases their palat-ability [ 104 106 ] and consumption [ 107 109 ]. Foodscontaining added glutamate are preferred to those without,even when the amount of Na ? is equivalent [ 110 ] and theuse of MSG has increased dramatically in recent decades[110 , 111 ]. MSG is found in signicant amounts in a widevariety of packaged foods ranging from fast-foods, snacks,dairy-based, conserved and dry vegetables, fat emulsions,dressings, processed meat, and poultry. MSG is also addedin basically unknown amounts in restaurant and industrialfood. Because food processors and manufacturers do nothave to list the amount of MSG on their packaging, it is noteasy to known how much MSG a person or child wouldingest in a day period.

Theoretically, the ability of MSG to enhance appetitemay cause hyperalimentation, obesity and, eventually,insulin resistance. However, whether or not MSG can causeobesity in humans remains controversial. In spite of theseveral efforts [ 112 , 113 ], there is lack of consensus on thisissue [ 73, 114 117 ]. In a recent nutrition survey, performedon 10,095 apparently healthy Chinese adults, the cumula-tive MSG intake was positively associated with BMI afteradjustment for potential confounders [ 116 ]. The adjusted

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hazard ratio of overweight was 1.33 (95% CI, 1.01, 1.75;P for trend \ 0.01) for participants in the highest quintile of MSG intake compared with those in the lowest quintileafter adjustment for age, physical activity, total energyintake, and other major lifestyle factors. Opposite resultswere reported by an analysis performed on 1,282 men andwomen who participated in the Jiangsu Nutrition Study,which concluded that when other food items or dietarypatterns were accounted for, no association existedbetween MSG intake and weight gain [ 115 ]. On the otherhand, this study has been criticized for serious methodo-logical aws [ 118 ].

While it is unlikely that glutamate derived from naturalunprocessed food would increase plasma glutamate con-centration to harmful levels, it has been shown that MSGingestion may induce signicant increases in plasma glu-tamate levels in both animals and humans. In piglet,increased glutamate concentrations were observed in theportal and arterial blood when the basal milk formula wassupplemented with MSG [ 119 ]. Also in larger pigs, tran-sient portal and arterial hyperglutamatemia was observedwhen the diet was supplemented with 10 g of MSG [ 120 ],indicating that large doses of glutamate may exceed theintestinal capacity to catabolize this amino acid. In men, theingestion of 150 mg/kg of MSG determines a rise in plasmaglutamate concentration of the 700800% and increasesintracellular free glutamate pool in the skeletal muscle formore than 1 h [ 121 ]. Plasma glutamate levels increasesignicantly and dose-dependently also in subjects ingest-ing MSG in consomme [122 ]. It should be also pointed outthat glutamate has signicant in vivo insulin-secretingeffects; humans who consume 10 g or more of MSG havedouble the normal plasma glutamate levels and higherinsulin concentrations [ 123 ]. In humans, an oral load of MSG determines a signicant increase in insulin secretionand insulin plasma levels [ 123 , 124 ]. The evidence thatingested MSG produces a direct effect on the secretoryfunction of the b -cell is notable and suggests that, similarlyto chronic hyperglycemia, persistent hyperglutamatemia (orchronically elevated extracellular glutamate levels in thepancreatic islets) may determine b -cell overstimulation andstress and even trigger b -cell autoimmunity.

Data regarding the role of maternal diet during preg-nancy, or infant feeding, in the development of b -cellautoimmunity are inconsistent. For instance, it has beenreported that maternal consumption of potatoes associateswith a signicant delay in the onset of b -cell autoantibodiesin the offspring, thereby suggesting a protective effect[125 ]. In contrast, two different studies have shown that inyoung children with increased susceptibility to T1D, earlyage at introduction of fruits, berries, and root vegetables(such as potatoes) was independently associated with b -cellautoimmunity [ 126 , 127 ]. The latter associations may

reect causal relationships and the question arises whethernatural or other toxins could be involved in the pathogen-esis of T1D [ 128 ]. It is puzzling, however, that an unknowncomponent of root vegetables could exert signicant, albeitopposite, effects on b -cell autoimmunity depending ontiming and route of administration. This evidence suggeststhat such component would be a normal constituent of thistype of food rather than a contaminant toxin. At this regard,it should be noted that root vegetables are particularly richin free glutamate [ 129 ] that remain stable during industrialprocessing [ 130 ]. Moreover, ingredients added to thesebaby foods during industrial processing or food preparationat home (i.e., MSG) could play a role. Besides, accordingto the accelerator hypothesis, the early introduction of supplementary foods may result in higher energy intake,which could cause b -cell stress and trigger b -cellautoimmunity.

Plasma glutamate levels in different pathologicconditions

Systemic plasma glutamate levels are elevated in severaldiseases characterized by chronic oxidative stress andinammation. Hyperglutamatemia has been found inobesity [ 131 ], liver diseases [ 132 , 133 ], cancer, and HIVinfection [ 134 136 ] even in the absence of symptoms of acquired immunodeciency syndrome [ 136 , 137 ]. In can-cer and HIV-infected persons, the increase in plasma glu-tamate has been related to the metabolic properties of thetumors combined with an altered glutamate metabolism[138 ]. Interestingly, in both these conditions, increasedglutamate levels are inversely correlated with lymphocyticactivity [ 136 ] and hyperglutamatemia has been implicatedin the pathogenesis of immunosuppression [ 136 138 ].Hyperglutamatemia is common in neurodegenerative dis-eases and in particular in stroke patients [ 139 141 ] where ithas been ascribed to an increased release of glutamate bythe activated platelets [ 139 , 140 ]. In multiple sclerosis,increased glutamate levels have been associated with amutation of the glutamate transporter GLT1 [ 142 ].Amyotrophic lateral sclerosis [ 143 145 ], Parkinsons dis-ease [ 146 ], epilepsy [ 147 ], autism [ 148 ], migraine [ 149 ],and depression [ 150 ] are also associated with increasedcirculating glutamate levels. Interestingly, hyperglutamat-emia has been shown also in patients with rheumatoidarthritis [ 151 ] where, as in stroke patients, it has beenrelated to the increased release of glutamate by the acti-vated platelets [ 139 , 140 ]. The relevance of the hyperglu-tamatemia observed under different pathologicalconditions to disease manifestation and natural history isnot always clear but indicates an important area of investigation.

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Although the causes responsible for the hyperglutamat-emia may vary in the different diseases, it is worth men-tioning that increased oxidative stress and inammation arecommon features of all these conditions and also of T1Dand T2D. Platelet activation is present in obesity, metabolicsyndrome, T1D, and T2D [ 152 155 ], and it might causehyperglutamatemia in these conditions. Stimulation of glutamate receptors consequent to platelet activation con-tributes to allograft rejection [ 156 ] and might as wellcontribute to the abnormal immunoregulation present inT1D. In addition, the pro-inammatory cytokine TNF- a ,commonly increased in obesity, insulin resistance, andother inammatory conditions, potentiates glutamate-induced cytotoxicity [ 157 ]. The synergic effects of inammation and hyperglutamatemia highlight the poten-tial contribution of the latter to diabetes pathogenesis.

Obesity is associated not only with insulin resistance of glucose and lipid metabolism [ 158 ] but also with proteinmetabolism [ 159 161 ]. Insulin is an anabolic hormone, andits action in suppressing breakdown and stimulating thesynthesis of proteins is impaired in insulin-resistant states.Protein and amino acid metabolism are abnormal in obese,insulin-resistant, and T2D subjects [ 162 166 ]. In obesity,defective protein metabolism is characterized by theimpaired ability of insulin to inhibit proteolysis, and sig-nicantly increased plasma glutamate levels have beendescribed in both obese and T2D subjects [ 162 , 167 , 168 ].Obesity is also associated with elevated plasmatic levels of leptin, a peptide hormone predominantly produced by theadipose tissue that circulates in the plasma in amountsproportional to body fat content [ 169 ]. A direct link hasbeen shown between leptin plasma levels and plateletactivation, and hyperleptinemia has been implicated in thepathogenesis of atherosclerosis in obesity [ 170 ]. The evi-dence that hyperleptinemia and hyperglutamatemia areboth present in obesity suggests a possible causative role of leptin-mediated platelet activation in increased glutamatelevels. It is also intriguing that leptin is a satiety factor thatacts on the same hypothalamic nuclei sensitive to MSGtoxicity [ 171 ] and that leptin can increase neuron excit-ability [ 172 ] and regulate the trafcking of NMDA andAMPA glutamate receptors [ 173 ]. Altogether, these evi-dences raise the exciting possibility that hyperleptinemiamight exert synergistic effects on glutamate-inducedcytotoxicity on both neural and non-neural tissues.

Increased plasmatic glutamate levels have been foundalso in insulin-resistant non-obese subjects [ 174 ] and ingestational diabetes [ 175 ]. It is noteworthy that in thesewomen, protein turnover was normalized by insulin treat-ment, but fasting and postprandial glutamate levelsremained elevated despite the satisfactory glycemic con-trol. There is clear evidence of altered protein metabolismalso in T1D [ 176 180 ]. In T1D, insulin deciency

increases protein breakdown and the associated hyperg-lucagonemia accelerates protein catabolism [ 181 ]. Analysisof serum metabolite proles between children who even-tually developed T1D and those who remained healthy andautoantibody-free showed a dramatic increase ( * 32-foldabove normal) in glutamate levels only in the children wholater developed T1D [ 182 ]. It is presently unknown howthese changes might have occurred and how higher levelsof circulating glutamate may interfere with T1D initiation.Theoretically, they could reect an increased glutamateoutput from the liver or muscle [ 167 ], but a dietary eventcannot be completely excluded. Whatever the cause, anincreased glutamate load in the b -cells might increase theactivity of GAD65, one of the major b-cell antigens [ 183 ,184 ]. High glutamate levels and increased GAD activitymay trigger or hasten autoimmunity and b -cell damage.Moreover, GAD-specic CD4 ? T cells may directly impairBBB function and induce an encephalomyelitis-like dis-ease [ 185 ]. The impaired BBB function might increase theload of glutamate in the CNS [ 185 ] and worsen neuro-toxicity that, in turn, might increase plasma glutamatelevels [ 139 147 ]. Hyperglutamatemia affects the immunesystem [ 136 138 , 186 ] and might also exert a suppressivefunction on regulatory T cells as glutamate decreasesInterleukin-10 secretion by peripheral blood lymphocytesof subjects with autoimmune thyroiditis [ 187 ]. Onepotential pathway linking high glutamate with theincreased risk of diabetes is the enhanced susceptibility of pancreatic b -cells to increased oxidative damage [ 188 ],which is an alternative mechanism of glutamate-inducedcytotoxicity [ 39, 189 ].

Interestingly, in the children who eventually developedT1D, glutamate levels were restored to normal after theappearance of the autoantibodies against GAD65 (GADA)or insulin (IAA) [ 182 ], raising the possibility that the initialautoimmune response is physiological and aimed atrestoring the metabolic homeostasis. According to thishypothesis, the disease may be caused or inuenced by adefective response toward the b -cell autoantigens [ 190 ,191 ]. Pancreatic b -cell cytoprotection due to diabetes-associated autoantibodies is indirectly supported by the factthat the autoantibody titers frequently decrease before theonset of clinical diabetes, which can be interpreted as asign of failing protection [ 192 ].

Pancreatic a -cells dysfunction may contributeto hyperglutamatemia

Pancreatic a -cells cosecrete glucagon together with gluta-mate that, in turn, modulates the secretion of glucagon,insulin, and somatostatin [ 42, 47, 193 195 ]. In humanislets, the glutamate released by the a -cells is a positive

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autocrine signal for glucagon secretion [ 194 ] and, conse-quently, for glutamate itself. a -Cell dysfunction andhyperactivity may thus trigger a vicious cycle that wouldmaintain and increase further the release of both glucagonand glutamate. The possible role of a -cells dysfunction inthe pathogenesis of diabetes, and in particular of the hy-perglutamatemia, is supported by a series of evidences.Plasma glucagon levels are abnormally elevated in bothT1D and T2D subjects, indicating that a-cell hypersecretionis a common diabetic feature [ 196 199 ]. Abnormally highfasting glucagon levels, suggestive of a -cell hypersecretion,have been found also in normoglycemic insulin-resistantobese adults and adolescents [ 200 204 ]. In the latter group,high fasting glucagon levels were observed in the face of elevated fasting insulin levels, suggesting that a -cellsmanifested insulin resistance to the suppressive effect of insulin on glucagon secretion. The correlation betweenbody weight and a -cell mass has never been studied indetail; however, a progressive increase in the a-cell number,leading to an imbalance between b- and a-cell mass, isexpected to occur in obesity. Interleukin-6 (IL6) is apleiotropic cytokine with metabolic effects [ 205 ], and itslevels are chronically elevated in obesity and predict thedevelopment of T2D [ 206 , 207 ]. IL6 is also a potent regu-lator of cellular proliferation [ 205 ], and the pancreatic a -cellis a primary target of IL6 function that increases glucagonsecretion and stimulates a -cell proliferation [ 208 ]. a -Cellmass expansion might also follow abnormal prohormoneconvertase 2 (PC2) activity. Pancreatic a -cells contain PC2and PC1/3 that act together to process proinsulin to insulin[209 211 ]. However, PC2 is also expressed by the a -cellsthat selectively converts proglucagon to glucagon [ 209 ,212 ]. Transgenic mice lacking PC2 activity have increasedlevels of intermediate forms of proinsulin in the circulation[209 , 210 , 213 ] but show also dramatic a -cell hypertrophyand hyperplasia [ 209 ] due to reduced a -cell death andincreased a-cell proliferation [ 214 ]. We have recently per-formed a retrospective analysis in a large baboon populationin which physical and clinical chemistry data, as well aspancreatic specimens, were available. We could thus dem-onstrate that islet amyloidosis was associated not only withincreased b -cell apoptosis and decreased relative b -cellvolume but also with signicant a -cell replication andhypertrophy and increased relative a-cell volume. Inbaboons, a -cell proliferation correlated with both hyperg-lucagonemia and hyperglycemia, providing new insightsinto the pathogenesis of the dysfunctional remodeling of the islet of Langerhans in this model of T2D [ 215 ].Unfortunately, we did not measure plasma glutamate levelsin those animals, and we do not know whether they werealso hyperglutamatemic. Nevertheless, the raising evidencethat a -cells dysregulation is present not only in diabetes, butalso in obesity and insulin resistance suggests that the

hyperglutamatemia described in these conditions [ 162 168 ,174 ] might be causally related also to a -cell hyperactivity.

Pancreatic b -cells are vulnerable to glutamate-inducedcytotoxicity

As glutamate receptors are widely expressed in severalorgans, their chronic supraphysiological stimulation maydetermine cytotoxicity also in non-neuronal tissues. Pan-creatic b -cells show numerous common features withneurons including shared transcriptional activators [ 216 ],the expression of proteins specialized in synaptic trans-mission, and the presence of neurotransmitters and theirreceptors [ 34, 217 , 218 ]. Islet cells express functionalglutamate receptors [ 45, 47, 219 , 220 ] and vesicular glu-tamate transporters [ 221 ], suggesting that glutamate is acrucial intercellular signal mediator. Moreover, since glu-tamate is cosecreted with glucagon, extracellular glutamateconcentrations might be particularly high in the pancreaticislet microenvironment.

Due to the many similarities between b -cells andneurons,we explored the possibility that high extracellular glutamatelevels would be toxic for the b -cells. In order to address thisissue, we performed in vitro studies on isolated human isletsand rodent b - and a-cells. Chronic exposure to glutamateinduced in human islets a dose-dependent increase in insulinsecretion under basal conditions that was paradoxicallyhigher than that under high glucose stimulation. Chronicexposure to glutamate also induced an increase in the pro-insulin-to-insulin ratio under both basal and stimulatedconditions [ 222 ]. All these secretory defects (increased basalinsulin secretion, reduced stimulated insulin release, andincreased proinsulin-to-insulin ratio) are typically observedin human islets damaged by chronic exposure to high glu-cose concentrations [ 223 225 ]. We also showed that a 3-dayexposure to glutamate induced a dose-dependent increase inhuman islet cell apoptosis that was statistically signicant at5 mmol/L glutamate and quantitatively similar to thatobserved at high glucose concentrations (16.7 mmol/L).Glutamate-induced apoptosis was restricted to the b -cells asconrmed by a quantitative electron microscopy analysis. Inthe absence of glutamate, the majority of a- (97 3%) andb -cells (78 1%) lacked nuclear and cytoplasmic degen-erations. Conversely, 75% of b -cells of islets exposed to5 mmol/L glutamate showed severe degenerative featuresincluding condensed apoptotic nuclei and numerous cyto-plasmic vacuoles, some of which contained dark bodies.Interestingly, a -cells of glutamate-exposed islets were wellpreserved.

In order to explore the mechanisms involved in gluta-mate toxicity in human islets, we performed experiments inthe presence of D-2-amino-5-phosphonovaleric acid and

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6-cyano-7-nitroquinoxaline-2,3-dione (CNQX), an AMPAand kainate receptor antagonist. We could thus demonstratethat CNQX does not prevent b -cell apoptosis induced bychronic incubation with glutamate as measured by bothELISA and electron microscopy. Moreover, consistent withprevious reports, the majority of glucose-responsive insu-lin-positive cells dispersed from isolated human islets didnot respond to glutamate with an increase in intracellularCa 2 ? . Oxidative stress is an alternative mechanisminvolved in glutamate-induced cytotoxicity [ 39], and ourdata actually suggest that this pathway is the prevalentmechanism of b -cell death in human islets. Originallydescribed in astrocytes, excess extracellular glutamatereverts the activity of the glutamate/cysteine antiportersystem x - c , thus depleting the cells of cysteine, a buildingblock of the antioxidant glutathione. In support of thishypothesis, we found that human islets expressed the x CT

subunit of the x - c exchanger. Furthermore, we foundincreased levels in adducts of 4-HNE (an irreversibleprotein modication product induced by oxidative stress[226 ] in lysates of human islets incubated in the presenceof 5 mmol/L glutamate for 3 days. This modication wasprevalently detected in b -cells as shown by double stainingwith insulin [ 222 ]. Interestingly, similar increase in adductsof 4-HNE was detected after a 3-day culture in 16.7 mmol/ L glucose, a recognized condition of oxidative stress inb -cells [ 222 ].

The likely elevated glutamate concentrations in the isletmicroenvironment, together with the demonstrated vul-nerability of human b -cells to the chronic exposure to highlevels of this amino acid, induced us to explore the pos-sibility that islets, as well as the CNS, would express aglutamate clearance system. We could thus demonstratethat the glutamate transporter GLT1 is present and func-tional in human islets and promotes b -cell survival [ 222 ].In both human and monkey pancreas, anti-GLT1 reactivitywas limited to the islets, whereas GLT1 protein was absentin the exocrine tissue. In both species, but even moreclearly in human, GLT1 staining was almost exclusivelylocalized to the cell membrane. Immunostaining withchromogranin, a marker of endocrine cells, conrmed thatGLT1 was expressed only in the islets and that its signalwas concentrated in the plasma membrane. Similar stainingat cellcell boundaries was detected in insulin-positivecells, conrming GLT1 expression by the b-cells. In con-trast, we did not observe colocalization of GLT1 with bothglucagon and somatostatin, suggesting that a- and d-cellsdo not express GLT1 or that if expressed it is under thelevel of detection. GLT1 is functional in isolated humanislets and is the main regulator of the glutamate clearancein the islets. In fact, selective GLT1 inhibition with di-hydrokainate (DHK) almost completely blocked the uptakeof glutamate in human islets and caused a dose-dependent

increase in apoptosis that was potentiated by the coappli-cation of glutamate [ 222 ]. Apoptosis was restricted tob -cells as shown by double staining for insulin and TUNELin dispersed islet cells [ 222 ].

The relevance of GLT1 to b -cell physiology was furtherinvestigated in murine b -cell line bTC3. Pharmacologicalinhibition of GLT1 activity with DHK signicantlyincreased the concentration of glutamate in the mediumand caused a parallel increase in b -cell apoptosis asrevealed by TUNEL assay [ 222 ]. Similar results wereobtained by downregulating the expression of the GLT1gene in bTC3 by means of a short hairpin RNA (shRNA).Interestingly, the shRNA construct increased bTC3 apop-tosis also in the absence of glutamate supplementation,suggesting that impaired GLT1 activity per se is suf-cient to induce bTC3 cell death. No effect was observed inthe presence of a control shRNA. These data conrm thatthe function of GLT1 is to control the extracellular gluta-mate concentration and preserve b -cell survival. To furthersupport this role of GLT1, we upregulated its expression bypharmacological treatment with cefriaxone (CEF). CEF is ab -lactam antibiotic that has been shown to increase brainGLT1 expression and activity and to induce neuroprotec-tion from glutamate toxicity in models of ischemic injuryand motor neuron degeneration. A 5-day incubation with10 and 100 l mol/L CEF induced a twofold increase inGLT1 expression also in bTC3 cells and led to a dose-dependent protection from glutamate-induced toxicity[222 ]. These data conrm that GLT1 is a key player in thecontrol of glutamate homeostasis and in the maintenance of b -cell integrity.

Pharmacological studies provide further evidence thatsusceptibility to glutamate toxicity is just another of themany similarities between b-cells and neurons. Essentially,all the antidiabetic drugs with known b -cell cytoprotectiveeffects such as GLP-1, exenatide, and glitazones [ 227 229 ]show also signicant neuroprotective activity [ 230 232 ]against glutamate-induced cytotoxicity [ 233 ]. Further,glitazones-mediated neuroprotection is associated withincreased GLT1 expression [ 234 , 235 ]. Therefore, pre-vention of glutamate toxicity may be an additional mech-anism by which these drugs exert their benecial effect onthe b-cells. Similar conclusions can be drawn by consid-ering the case of topiramate, an antiepileptic agent thatprovides neuroprotection by counteracting glutamate tox-icity [ 236 , 237 ] but which has also signicant antidiabeticand b-cell cytoprotective effects [ 238 241 ]. In rodentmodels of T2D, topiramate improves glucose-stimulatedinsulin release and increases islet insulin content [ 242 ], andin vitro exposure of rodent b -cells to topiramate preventslipotoxicity [ 243 ]. Incidentally, we recently describedlong-lasting remission ( [ of 5 years) of T1D after treat-ment with topiramate for generalized seizures [ 244 ].

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Altogether, these data suggest that drugs that target gluta-mate toxicity may be helpful against both neurodegenera-tion and diabetes.

Concluding remarks

Abnormal glutamate homeostasis is commonly observed indiabetes, obesity, and insulin resistance and may beinvolved in pancreatic b -cell loss. This is particularly rel-evant in view of the accelerator hypothesis that con-siders T1D and T2D the same disease, set on differentgenetic backgrounds, and where insulin resistance acts asmajor link. According to this hypothesis, the rst acceler-ator is an intrinsic b -cell fragility necessary for the devel-opment of diabetes but insufcient per se to cause it, thesecond accelerator is insulin resistance resulting fromweight gain and leading to b -cell overstimulation and stressand, nally, the third accelerator is the development of b -cell autoimmunity in a minority of genetically predis-posed subjects.

The hyperglutamatemia described in diabetic and obesesubjects is generally ascribed to abnormal protein metab-olism in liver and muscle and excessive glutamate releaseby activated platelets. However, accumulating data in favorof the presence of a -cell dysfunction and hypersecretion indiabetes, obesity, and insulin resistance, together with theevidence that glutamate is cosecreted with glucagon, maycontribute to the hyperglutamatemia described in theseconditions. We recently showed that high extracellularglutamate levels are toxic for human b-cells and thatmaintenance of a normal glutamate homeostasis is criticalto preserve b-cell integrity. We also showed that the glu-tamate transporter GLT1 is expressed on the b-cell mem-brane and, by controlling extracellular glutamate levels inthe islet microenvironment, plays a pivotal role in theseprocesses. Hyperglutamatemia might be thus considered anovel b-cell insult, and its toxic effects may be moredevastating on genetically fragile b-cells. Moreover, ashyperglutamatemia impacts on the immune system, ingenetically predisposed subjects, it might also foster b -cellautoimmunity.

Excessive glutamate consumption with food, mainly inthe form of MSG supplementation, may cause obesity andinsulin resistance. The use of MSG has increased inrecent years, and MSG is found in signicant amounts ina wide variety of foods consumed habitually also by veryyoung children. It is presently unclear whether, undercertain circumstances, glutamate-enriched foods mayactually increase plasma glutamate to toxic levels. Themost critical scenario would be a breast-fed newborn of amother consuming a MSG-enriched diet, where thepotential increases in mothers plasma glutamate levels

would be amplied in the milk by a factor of 40 byconsidering the exceptional capability of the mammarygland to concentrate glutamate. If further studies willundoubtedly conrm that exaggerate MSG intake inducesobesity, the impact of glutamate on the second, mostimportant, accelerator will then be denitely proved.Increased weight gain is a recognized risk factor not onlyfor T2D but also for T1D in young children [ 1921, 245 ,246 ]. By inducing insulin resistance, as well as bydirectly stimulating insulin secretion, excessive glutamateloads would also impact on the other accelerators andwould determine b -cell overstimulation, which maydecompensate an intrinsically fragile b-cell populationand may as well increase the expression of b -cell anti-gens. Moreover, the evidence that leptin has signicanteffects on platelet activation, glutamate-induced excit-ability, and glutamate receptor turnover raise the possi-bility that obesity-associated hyperleptinemia mightcontribute to the genesis of hyperglutamatemia and mightplay synergistic effects on glutamate-induced cytotoxicityon pancreatic b-cells.

There is growing evidence that glutamate toxicity hasalso peripheral neural and non-neural tissue targets. A largequantity of ingested glutamate may induce enteric excito-toxicity and cause the increased intestinal permeabilityobserved early in diabetes. In turn, augmented entericpermeability may increase the absorption of free glutamatefrom the intestinal lumen to the blood stream, therebycontributing further to the hyperglutamatemia described indiabetic subjects. Of course, increased intestinal perme-ability might also challenge the immune system withenvironmental diabetogenic antigens of viral and alimen-tary origin.

We conclude this short review article suggesting that thehyperglutamatemia, whose generation can involve thecontribution of insulin resistance, a -cell dysfunction,platelet activation, and abnormal glutamate metabolism inliver and muscle, may play an important, yet unrecognized,role in the progressive b -cell loss occurring in T1D andT2D. In line with the accelerator hypothesis, and whenconsidering the b -cell vulnerability to glutamate toxicity,endogenous pathogenic hyperglutamatemia and excessiveMSG ingestion with food might contribute to diabetesepidemic nowadays. Lastly, the evidence that high extra-cellular glutamate concentrations and abnormal GLT1function may cause b -cell death would open new avenuesin the search of novel pharmacological approaches fordiabetes treatment and prevention.

Acknowledgments This work was supported by UniversityResearch Program 2008 (to CP) and National Institutes of HealthGrant RO1 DK080148 (to FF). AMD, CP, and FF and Eliana Sara diCairano (ESDC) are inventors in a Patent Cooperation Treaty appli-cation (PCT/EP09/08256, US2011/0244486A1).

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