Aldose Reductase and the Polyol Pathway in Schwann Cells: Old and New Problems

International Journal of Molecular Sciences

Review Aldose Reductase and the Polyol Pathway in Schwann Cells: Old and New Problems

Naoko Niimi 1,*, Hideji Yako 1 , Shizuka Takaku 1, Sookja K. Chung 2 and Kazunori Sango 1

1 Diabetic Neuropathy Project, Tokyo Metropolitan Institute of Medical Science, Tokyo 156-8506, Japan; [email protected] (H.Y.); [email protected] (S.T.); [email protected] (K.S.) 2 Medical Faculty, Macau University of Science and Technology, Macau 999078, China; [email protected] * Correspondence: [email protected]; Tel.: +81-3-6834-2359; Fax: +81-5316-3150

Abstract: Aldose reductase (AR) is a member of the reduced nicotinamide adenosine dinucleotide phosphate (NADPH)-dependent aldo-keto reductase superfamily. It is also the rate-limiting enzyme of the polyol pathway, catalyzing the conversion of glucose to sorbitol, which is subsequently converted to fructose by sorbitol dehydrogenase. AR is highly expressed by Schwann cells in the peripheral nervous system (PNS). The excess glucose flux through AR of the polyol pathway under hyperglycemic conditions has been suggested to play a critical role in the development and progression of diabetic peripheral neuropathy (DPN). Despite the intensive basic and clinical studies over the past four decades, the significance of AR over-activation as the pathogenic mechanism of DPN remains to be elucidated. Moreover, the expected efficacy of some AR inhibitors in patients with DPN has been unsatisfactory, which prompted us to further investigate and review the understanding of the physiological and pathological roles of AR in the PNS. Particularly, the investigation of AR and the

polyol pathway using immortalized Schwann cells established from normal and AR-deficient mice could shed light on the causal relationship between the metabolic abnormalities of Schwann cells and

Citation: Niimi, N.; Yako, H.; discordance of axon-Schwann cell interplay in DPN, and led to the development of better therapeutic Takaku, S.; Chung, S.K.; Sango, K. strategies against DPN. Aldose Reductase and the Polyol Pathway in Schwann Cells: Old and Keywords: Schwann cells; aldose reductase; polyol pathway; hyperglycemic conditions; immortal- New Problems. Int. J. Mol. Sci. 2021, ized Schwann cell lines; aldehyde detoxiﬁcation 22, 1031. https://doi.org/10.3390/ ijms22031031

Academic Editor: 1. Introduction Mariarosaria Santillo In the peripheral nervous system (PNS), blood glucose is incorporated into cells via Received: 11 December 2020 glucose transporters (GLUT 1 and GLUT 3) in an insulin-independent manner. Under nor- Accepted: 19 January 2021 Published: 21 January 2021 moglycemic conditions, most of the cellular glucose is converted to glucose 6-phosphate by hexokinase and further metabolized into pyruvate through the glycolytic pathway.

Publisher’s Note: MDPI stays neutral However, under hyperglycemic conditions, the saturation of the glycolytic pathway esca- with regard to jurisdictional claims in lates the flux of glucose through the polyol pathway, the first and major collateral route published maps and institutional affil- of glucose metabolism. The polyol pathway, which initially identifies in the seminal vesi- iations. cle [1], consists of the reduction step of glucose to sorbitol and the oxidation step of sorbitol to fructose: the first reduction step is catalyzed by aldose reductase (AR) in a reduced nicotinamide adenosine dinucleotide phosphate (NADPH)-dependent manner, and the second oxidation step is catalyzed by sorbitol dehydrogenase (SDH) in an NAD-dependent manner, respectively (Figure1). Polyol pathway hyperactivity induces multiple metabolic Copyright: © 2021 by the authors. Licensee MDPI, Basel, Switzerland. imbalances, described in the following sections, and is thereby considered as a major cause This article is an open access article of diabetic peripheral neuropathy (DPN) [2], as well as other complications [3,4]. AR, being distributed under the terms and the rate-limiting enzyme of the polyol pathway, has been studied intensively as a target conditions of the Creative Commons protein for the prevention and treatment of DPN. Although numerous AR inhibitors (ARI) Attribution (CC BY) license (https:// have been developed with promising results in the cell and animal models of DPN, most creativecommons.org/licenses/by/ of the clinical trials using them ended in failure due to the adverse effects and/or limited 4.0/). drug potency [5]. Currently, epalrestat (Table1) is the only ARI available for clinical use in

Int. J. Mol. Sci. 2021, 22, 1031. https://doi.org/10.3390/ijms22031031 https://www.mdpi.com/journal/ijms Int. J. Mol. Sci. 2021, 22, x FOR PEER REVIEW 2 of 16

ous AR inhibitors (ARI) have been developed with promising results in the cell and animal models of DPN, most of the clinical trials using them ended in failure due to the adverse effects and/or limited drug potency [5]. Currently, epalrestat (Table 1) is the only ARI available for clinical use in Japan, but its use is limited to the patients with DPN at early stages and stable glycemic control [6]. Despite the intensive studies over the past four decades, the significance of AR and the polyol pathway in the pathogenesis of DPN and effective therapies against it remains unknown. AR is predominantly localized at Schwann cells in the PNS [7], and its activation and subsequent sorbitol accumulation in Schwann cells under hyperglycemic conditions appear to play a critical role in the development and progression of DPN [8]. Histopatholog- ical studies on peripheral nerve specimens from patients and experimental animals with DPN indicated the vulnerability of Schwann cells and its close association with nerve dysfunction, such as reduced nerve conduction velocity (NCV), decreased sensation, spontaneous pain, and impaired axonal regeneration [9]. In agreement with these findings, transgenic mice overexpressing human AR in Schwann cells displayed more severe neurological abnormalities than non-transgenic littermate mice under hyperglycemic conditions [10]. In contrast, AR-deficient mice created by gene manipulation showed a defense against neurological manifestations upon inducing diabetes [11]. However, the precise causal relationship between the AR/polyol pathway in Schwann cells and the pathogene- Int. J. Mol. Sci. 2021, 22, 1031 sis of DPN remains unclear. Recent studies using AR-deficient mice [12] and Schwann2 of 14 cells [13] have raised new issues, such as crosstalk among the polyol and other collateral glucose-utilizing pathways. In this paper, we overview the previous studies regarding the rolesJapan, of AR but in its Schwann use is limited cells to under the patients diabetic with and DPN non-diabetic at early stages conditions, and stable and glycemic then we introducecontrol [immortalized6]. Despite the Schwann intensive cells, studies established over the past in our four laboratory decades, the[13–16], signiﬁcance as valuable of toolsAR for and the the study polyol of pathway DPN, focusing in the pathogenesis particularly of on DPN AR and effectivethe polyol therapies pathway. against it remains unknown.

Figure 1. Glucose metabolism and the polyol pathway in the peripheral nervous system. Under normoglycemic conditions Int.Figure J.Int. Mol. J.Int. 1. Mol.Sci. Glucose J. Mol. 2021Sci. 2021Sci., 22 metabolism ,2021 ,x 22 FOR, ,x 22 FOR PEER, x FOR PEER and REVIEW PEER REVIEWthe REVIEWpolyol pathway in the peripheral nervous system. Under normoglycemic conditions3 of 316 of 3 16of 16 (a), most of the glucose incorporated into the cells via glucose transporters (GLUT) is metabolized into pyruvate through the (a), most of the glucose incorporated into the cells via glucose transporters (GLUT) is metabolized into pyruvate through the glycolyticpathway, pathway, and and less less than than 3% 3% of theof the glucose glucose enters enters the polyolthe polyol pathway. pathway. However, Howe underver, hyperglycemicunder hyperglycemic conditions conditions(b), the(b), escalationthe escalation of glucose of glucose flux flux into theinto polyol the polyol pathway pathway results results in the in accumulation the accumulation of sorbitol of sorbitol and fructose and fructose and the and TableTable 1.Table Aldose 1. Aldose 1. Aldosereductase reductase reductase inhibitors+ inhibitors inhibitors described described described in this in thisarticle. in this article. article. the consumption ofof cofactorscofactorsof ofAR AR (NADPH) (NADPH) and and SDH SDH (NAD (NAD).+). IncreasedIncreased sorbitol sorbitol contents contents lead lead to to the the depletion depletion of of myo- myo- inositolinositol and and taurine, taurine, and and reduced reducedEpalrestatEpalrestat myo-inositol myo-inositolEpalrestat contents cancan be be a a cause FidarestatcauseFidarestat of ofFidarestat decreases decreases in phosphoinositidein phosphoinositide andRanirestat Ranirestat diacylglycerol.andRanirestat diacylglyc- NameName Name erol.Red Red and and blue blue arrows arrows indicate indicate(Kinedak increases(Kinedak increases(Kinedak) and) and decreases) decreases in the in amount the(SNK-860) amount(SNK-860) of molecules,(SNK-860) of molecules, respectively. respectively. (AS-3201)(AS-3201)(AS-3201) MolecularMolecularMolecular For- For- For- C15HCTable1315NOHC1315NOH 1.3S132Aldose NO3S2 3S2 reductase inhibitorsC12H describedC1012FNHC10123FNHO104 in3FNO this4 3O4 article. C17HC1117BrFNHC1117BrFNH113OBrFN4 3O4 3O4 mulamula mula MolecularMolecularMolecular Epalrestat Fidarestat Ranirestat Name 319.4319.4 319.4 ® 279.2279.2 279.2 420.2 420.2 420.2 WeightWeightWeight (Kinedak ) (SNK-860) (AS-3201) InternationalMolecularInternationalInternational Formula C15H13NO3S2 C12H10FN3O4 (3R)-2’-[(4-bromo-2-fluoro-(3R)-2’-[(4-bromo-2-fluoro-(3R)-2’-[(4-bromo-2-fluoro-C17H11BrFN3O4 2-[(5Z)-5-[(E)-2-methyl-3-phe-2-[(5Z)-5-[(E)-2-methyl-3-phe-2-[(5Z)-5-[(E)-2-methyl-3-phe- UnionMolecularUnion Unionof Pure of Weight Pure of Pure 319.4(2S,4S)-6-fluoro-2’,5’-dioxospiro[2(2S,4S)-6-fluoro-2’,5’-dioxospiro[2(2S,4S)-6-fluoro-2’,5’-dioxospiro[2 279.2 phenyl)methyl]phenyl)methyl] 420.2phenyl)methyl] nylprop-2-enylidene]-4-oxo-2-nylprop-2-enylidene]-4-oxo-2-nylprop-2-enylidene]-4-oxo-2-2-[(5Z)-5-[(E)-2-methyl-3- (3R)-2’-[(4-bromo-2- Internationalandand Appliedand Applied Union Applied of Pure ,3-dihydrochromene-4,4’-imidazol-,3-dihydrochromene-4,4’-imidazol-,3-dihydrochromene-4,4’-imidazol-(2S,4S)-6-fluoro-2’,5’-dioxospiro[2 spiro[pyrrolidine-3,4’-pyr-spiro[pyrrolidine-3,4’-pyr-spiro[pyrrolidine-3,4’-pyr- phenylprop-2-enylidene]-4-oxo-2- fluorophenyl)methyl] and Applied Chemistrysulfanylidene-1,3-thiazolidin-3-sulfanylidene-1,3-thiazolidin-3-sulfanylidene-1,3-thiazolidin-3- ,3-dihydrochromene-4,4’- ChemistryChemistryChemistry sulfanylidene-1,3-thiazolidin-3- idine]-2-carboxamideidine]-2-carboxamideidine]-2-carboxamide rolo[1,2-a]pyrazine]-1’,2,3’,5-tet-spiro[pyrrolidine-3,4’-pyrrolo[1,2-rolo[1,2-a]pyrazine]-1’,2,3’,5-tet-rolo[1,2-a]pyrazine]-1’,2,3’,5-tet- (IUPAC) Name yl]aceticyl]aceticyl]acetic acid acid acid imidazolidine]-2-carboxamide (IUPAC)(IUPAC)(IUPAC) Name Name Name yl]acetic acid a]pyrazine]-1’,2,3’,5-tetroneronerone rone

ChemicalChemicalChemical Struc- Struc- Struc- Chemical Structure tureture ture

Current Status It isIt commercially isItIt commercially isis commercially commercially available availableavailable available in in Japanin in Its development was terminated Its development was terminated CurrentCurrentCurrent Status Status Status Its developmentIts developmentIts development was was terminated was terminated terminated Its development Its development Its development was was terminated was terminated terminated JapanJapan Japan AR is predominantly localized at Schwann cells in the PNS [7], and its activation and subsequent2. Physiological2. Physiological2. Physiological sorbitol and accumulation and Pathological and Pathological Pathological in Roles Schwann Roles ofRoles AR of cells ARinof under SchwannAR in Schwann in hyperglycemic Schwann Cells Cells Cells conditions appear to2.1. play 2.1.Possible2.1. aPossible critical Possible Functions Functions role Functions in of theAR of development ARin of Schwan ARin Schwan in Schwann Cellsn and Cells nunder progressionCells under Non-Diabetic under Non-Diabetic Non-Diabetic of DPN Conditions [Conditions8]. Conditions Histopathological studiesThe onThe aldo-keto peripheralThe aldo-keto aldo-keto reductase nerve reductase reductase specimens (AKR) (AKR) (AKR)superfamily from superfamily patientssuperfamily consists and consists experimentalco ofnsists 15 of family15 of family15 animals familygroups groups groups with(AKR1-15) (AKR1-15) DPN (AKR1-15) indicatedwithwith variouswith various the various vulnerabilitykinds kinds ofkinds NADP(H)-dependentof ofNADP(H)-dependentof Schwann NADP(H)-dependent cells and ox its idoreductasesox close idoreductasesoxidoreductases association [17]. [17]. with AR [17]. AR nervebelongs ARbelongs dysfunc-belongs to theto theto the tion,AKR1BAKR1B suchAKR1B subgroup as subgroup reduced subgroup of the nerve of theAKR1 of conduction theAKR1 familyAKR1 family family(human velocity (human (human and (NCV), and murine and murine decreased murine AR ARare ARsensation,aretermed aretermed termed as spontaneousAKR1B1 as AKR1B1 as AKR1B1 and and and pain,AKR1B3,AKR1B3, andAKR1B3, impairedrespectively), respectively), respectively), axonal and regeneration and the and thesubgroup thesubgroup subgroup [9]. is In characterized agreementis characterized is characterized with by thesereducingby byreducing findings, reducing saccharides saccharides transgenic saccharides and and and mice overexpressing human AR in Schwann cells displayed more severe neurological aldehydesaldehydesaldehydes derived derived derived from from lipid from lipid peroxidation, lipid peroxidation, peroxidation, steroids, steroids, steroids, and and their and their derivatives their derivatives derivatives [18]. [18]. As [18]. com-As Ascom- com- abnormalities than non-transgenic littermate mice under hyperglycemic conditions [10]. paredpared withpared with other with other AKR1B other AKR1B AKR1B constituents, constituents, constituents, AR ARseem ARseems toseems be to s morebe to morebe ubiquitously more ubiquitously ubiquitously expressed expressed expressed [19– [19– [19– In contrast, AR-deficient mice created by gene manipulation showed a defense against 22],22], with22], with predominant with predominant predominant localization localization localization at the at the cytoplasmat thecytoplasm cytoplasm of the of the targetof thetarget tissuestarget tissues tissuesof diabetic of diabetic of diabetic com- com- com- neurological manifestations upon inducing diabetes [11]. However, the precise causal plications,plications,plications, such such as such theas thevasculature,as thevasculature, vasculature, PNS PNS (particularly PNS (particularly (particularly Schwann Schwann Schwann cells), cells), eyes,cells), eyes, and eyes, and kidneys and kidneys kidneys relationship between the AR/polyol pathway in Schwann cells and the pathogenesis of [23].[23]. AR[23]. ARhas AR hasbeen has been receiving been receiving receiving much much attentionmuch attention attention as a as possible aas possible a possible culprit culprit culpritof DPN of DPNof and DPN and other and other com- other com- com- DPN remains unclear. Recent studies using AR-deficient mice [12] and Schwann cells [13] plicationsplicationsplications over over the over thepast thepast four past four decades, four decades, decades, whereas whereas whereas its physiologicalits physiologicalits physiological roles roles remain roles remain remain largely largely largely un- un- un- have raised new issues, such as crosstalk among the polyol and other collateral glucose- known.known.known. AR ARseems ARseems toseems be to involvedbe to involvedbe involved in the in the maintenancein themaintenance maintenance of cellular of cellular of cellular homeostasis homeostasis homeostasis through through through os- os- os- utilizing pathways. In this paper, we overview the previous studies regarding the roles of moticmotic regulationmotic regulation regulation and and detoxification. and detoxification. detoxification.

2.1.1.2.1.1. Responses2.1.1. Responses Responses to Hyperosmotic to Hyperosmotic to Hyperosmotic Stress Stress Stress In theIn thekidney,In thekidney, kidney, hyperosmotic hyperosmotic hyperosmotic stress stress increasesstress increases increases the theAR the ARactivity ARactivity activity and and the and theamount theamount amount of sor- of sor- of sorbitol,bitol, whichbitol, which balanceswhich balances balances the theosmotic theosmotic osmotic pressure pressure pressure of extracellular of extracellular of extracellular sodium sodium sodium chloride chloride chloride (NaCl) (NaCl) (NaCl) [24]. [24]. In [24]. In In additionadditionaddition to the to the osmoregulation,to theosmoregulation, osmoregulation, AR ARmay ARmay be may essentialbe essentialbe essential for forthe forthematuration thematuration maturation of the of the urineof theurine con- urine con- con- centratingcentratingcentrating mechanism mechanism mechanism [25,26]. [25,26]. [25,26]. The The augmented The augmented augmented flux flux into flux into the into thepolyol thepolyol polyolpathway pathway pathway under under expo-under expo- expo- suresure tosure hypertonicto hypertonicto hypertonic conditions conditions conditions has hasbeen has been sh beenown shown shinown manyin manyin othermany other cells,other cells, including cells, including including Schwann Schwann Schwann cellscells [27–29];cells [27–29]; [27–29]; however, however, however, the thePNS thePNS is PNSusually is usually is usually unaffected unaffected unaffected by extracellularby extracellularby extracellular osmotic osmotic osmotic stress stress andstress and and the theosmoregulatory theosmoregulatory osmoregulatory role role of rolethe of the AR/polyolof theAR/polyol AR/polyol pathway pathway pathway in Schwann in Schwann in Schwann cells cells remains cells remains remains obscure. obscure. obscure.

2.1.2.2.1.2. Aldehyde2.1.2. Aldehyde Aldehyde Detoxification Detoxification Detoxification AR ARand ARand other and other AKRs other AKRs areAKRs areinvolved areinvolved involved in the in thedeto in thedetoxification detoxificationxification of reactive of reactive of reactive biogenic biogenic biogenic aldehydes, aldehydes, aldehydes, suchsuch assuch 4-hydroxy-2-nonenalas 4-hydroxy-2-nonenalas 4-hydroxy-2-nonenal (4HNE), (4HNE), (4HNE), acrolein, acrolein, acrolein, methylglyoxal methylglyoxal methylglyoxal (MG), (MG), (MG),and and 3-deoxyglu- and 3-deoxyglu- 3-deoxyglu- cosonecosonecosone (3-DG) (3-DG) (3-DG) [30]. [30]. Although [30]. Although Although AR ARhas AR hasbeen has been sugg been suggested suggested asested an as efficient an as efficientan efficient catalyst catalyst catalyst for thefor forthereduc- thereduc- reduc- tiontion of tionthese of theseof substances these substances substances [31,32], [31,32], [31,32], neither neither neither AR ARgene ARgene ablation gene ablation ablation nor norARI nor ARI treatment ARI treatment treatment augmented augmented augmented theirtheir toxicitytheir toxicity toxicity against against against Schwann Schwann Schwann cells cells [13,33]. cells [13,33]. [13,33]. These These findingsThese findings findings suggest suggest suggest the thecomplex thecomplex complex metabolic metabolic metabolic disposaldisposaldisposal system system system of reactive of reactive of reactive aldehydes, aldehydes, aldehydes, wh ichwh ichwhwill ichwill be will fullybe fullybe discussed fully discussed discussed in Section in Section in Section 4. 4. 4.

Int. J. Mol. Sci. 2021, 22, 1031 3 of 14

AR in Schwann cells under diabetic and non-diabetic conditions, and then we introduce immortalized Schwann cells, established in our laboratory [13–16], as valuable tools for the study of DPN, focusing particularly on AR and the polyol pathway.

2. Physiological and Pathological Roles of AR in Schwann Cells 2.1. Possible Functions of AR in Schwann Cells under Non-Diabetic Conditions The aldo-keto reductase (AKR) superfamily consists of 15 family groups (AKR1-15) with various kinds of NADP(H)-dependent oxidoreductases [17]. AR belongs to the AKR1B subgroup of the AKR1 family (human and murine AR are termed as AKR1B1 and AKR1B3, respectively), and the subgroup is characterized by reducing saccharides and aldehydes derived from lipid peroxidation, steroids, and their derivatives [18]. As compared with other AKR1B constituents, AR seems to be more ubiquitously expressed [19–22], with predominant localization at the cytoplasm of the target tissues of diabetic complications, such as the vasculature, PNS (particularly Schwann cells), eyes, and kidneys [23]. AR has been receiving much attention as a possible culprit of DPN and other complications over the past four decades, whereas its physiological roles remain largely unknown. AR seems to be involved in the maintenance of cellular homeostasis through osmotic regulation and detoxification.

2.1.1. Responses to Hyperosmotic Stress In the kidney, hyperosmotic stress increases the AR activity and the amount of sorbitol, which balances the osmotic pressure of extracellular sodium chloride (NaCl) [24]. In addition to the osmoregulation, AR may be essential for the maturation of the urine concentrating mechanism [25,26]. The augmented flux into the polyol pathway under exposure to hypertonic conditions has been shown in many other cells, including Schwann cells [27–29]; however, the PNS is usually unaffected by extracellular osmotic stress and the osmoregulatory role of the AR/polyol pathway in Schwann cells remains obscure.

2.1.2. Aldehyde Detoxification AR and other AKRs are involved in the detoxification of reactive biogenic aldehydes, such as 4-hydroxy-2-nonenal (4HNE), acrolein, methylglyoxal (MG), and 3-deoxyglucosone (3-DG) [30]. Although AR has been suggested as an efficient catalyst for the reduction of these substances [31,32], neither AR gene ablation nor ARI treatment augmented their toxicity against Schwann cells [13,33]. These findings suggest the complex metabolic disposal system of reactive aldehydes, which will be fully discussed in Section4.

2.1.3. Steroid Metabolism Neuroactive steroids, such as progesterone, testosterone and their reduced metabolites, have been shown to promote Schwann cell proliferation and myelin protein synthesis [34] and restore DPN through regulating myelin lipid proﬁles [35]. Schwann cells not only uptake these steroids via steroid receptors, but also produce and metabolize them [34]. AR is recognized to catalyze the reduction of progesterone and its related steroids [4], but whether their metabolism is affected by the polyol pathway hyperactivity under diabetic conditions or AR inhibition remains to be determined [36].

2.2. The Polyol Pathway as a Major Pathogenic Factor of DPN Current evidence suggests that polyol pathway hyperactivity in the PNS (especially Schwann cells) under diabetic conditions triggers multiple metabolic abnormalities associated with DPN.

2.2.1. Increased Sorbitol Contents Because sorbitol is a membrane-impermeable substance, its accumulation was thought to impose osmotic stress on Schwann cells and the PNS (“Osmotic Hypothesis”). However, sorbitol concentrations under hyperglycemic conditions were found to be osmotically irrelevant, and the extracellular sorbitol level was signiﬁcantly increased as compared Int. J. Mol. Sci. 2021, 22, 1031 4 of 14

with that under normoglycemic conditions in primary cultured Schwann cells [28]. These findings suggest that sorbitol can be released from Schwann cells by an unidentified transport mechanism [37], and the sorbitol-induced osmotic stress is unlikely to perturb Schwann cell function [38]. Rather, the increase in sorbitol contents leads to the depletion of other osmolytes, such as myo-inositol and taurine [39], whose changes may be more relevant to the pathogenesis of DPN (Figure1). Because myo-inositol is an important constituent of the phospholipids, its depletion leads to a decrease in phosphoinositide and diacylglycerol contents, which, in turn, diminishes the activity of protein kinase C (PKC) and Na+–K+-ATPase [40–42]. Taurine works as an antioxidant, calcium modulator, and neurotransmitter, and its decrease has been suggested to cause oxidative and nitrosative stress in Schwann cells [43]. Although no studies addressed the efficacy of taurine in patients with DPN, its neuropharmacological potential is attracting attention [44].

2.2.2. Redox State Changes The polyol pathway hyperactivity and consequent consumption of cofactors of AR (NADPH) and SDH (NAD+) trigger a cascade of interrelated metabolic imbalances (Figure2). NADPH is a common cofactor of AR, nitric oxide synthase (NOS), and glutathione reductase (GR), and its depletion due to the increase in AR activity leads to the inhibition of the other enzymes [45]. The reduced NOS activity and NO contents can be a cause of diminished nerve blood flow, whereas the decreases in reduced glutathione (GSH) by GR inhibition results in the excessive production of free radicals and the enhancement of oxidative stress [46]. During the second step of the pathway catalyzed by SDH, NAD+ is converted to the reduced form NADH. Because NADH is a substrate for NADH oxidase, the conversion leads to the upregulation of NADH oxidase activity and the production of superoxide anions, thereby contributing to Int. J. Mol. Sci. 2021, 22, x FOR PEER REVIEWoxidative stress [47]. Moreover, the NAD+/NADH redox state imbalances may be involved5 of 16 in

the activation of PKCβ, which also plays a role in the development of DPN [2,48]. Regarding Schwann cells, however, direct evidence to prove these causal dependences is lacking.

FigureFigure 2. 2.RedoxRedox state state changes changes induced induced by bythe thepolyol polyol pathway pathway hyperactivity hyperactivity under under high high glucose glucose conditions.conditions. Nicotinomide Nicotinomide adenoside adenoside dinucleotide dinucleotide phosphat phosphatee (NADPH) consumption duringduring thethe ﬁrst firststep step catalyzed catalyzed by aldoseby aldose reductase reductase (AR) (AR) suppresses suppresses the the activities activities of glutathione of glutathione reductase reductase (GR) and (GR)nitric and oxide nitric synthase oxide synthase (NOS), which (NOS), leads which tothe leads reduction to the reduction in glutathione in glutathione (GSH) and (GSH) NO,respectively. and NO, respectively.NADH upregulation NADH upregulation during the during second the step second catalyzed step bycatalyzed SDH increases by SDH theincreases activities the ofactivi- NADH β tiesoxidase of NADH and PKCoxidaseβ. GAP: and PKC glyceraldehyde. GAP: glyceraldehyde 3-phosphate, 3-phosphate, DHAP: dihydroxyacetone DHAP: dihydroxyacetone phosphate. Red phosphate.and blue arrows Red and indicate blue arrows increases indicate and decreases increases in and the decreases amount or in activity the amount of molecules, or activity respectively. of molecules, respectively. 2.2.3. Formation of Dicarbonyl Compounds and Advanced Glycation Endproducts (AGEs) 2.2.3. FormationAs the second of Dicarbonyl step of the Compounds polyol pathway, and Advanced sorbitol is Glycation converted Endproducts to fructose by SDH. (AGEs)Fructose is further metabolized into dicarbonyl compounds such as 3-deoxyglucose (3-DG) andAs methylglyoxal the second (MG),step of which the polyol are recognized pathway, as sorbitol potent glycating is converted agents to and fructose participate by SDH. in the Fructoseformation is offurther AGEs metabolized [49,50]. Besides into the dicarbonyl AGEs induced compounds Schwann cellsuch injury as 3-deoxyglucose and death [51,52 (3-], 3- DG) and methylglyoxal (MG), which are recognized as potent glycating agents and participate in the formation of AGEs [49,50]. Besides the AGEs induced Schwann cell injury and death [51,52], 3-DG and MG exhibit direct toxicity toward Schwann cells [33,53,54]. Recent studies suggest the formation of AGEs from exogenous (diet-derived) and endogenous (polyol-pathway-derived) fructose, and the fructose-derived AGEs have been attracting attention as a novel pathogenic factor for a variety of metabolic, inflammatory, and neurodegenerative diseases [55,56].

2.2.4. PKC Activity Abnormalities Hyperglycemic insults have been shown to affect PKC activity in a cell- and isozyme- specific manner; PKCα activity is reduced in Schwann cells, whereas PKCβ activity is increased in vascular endothelial and smooth muscle cells [2,57]. These findings led us to suppose the involvement of α isozyme inactivation in DPN and β isozyme activation in microangiopathy (including reduced nerve blood flow), respectively. AR/polyol pathway is suggested to interact with PKC signaling cascades and differentially influence the expression/activity of the α and β isozymes [48,58]. As described above, myo-inositol depletion can be a cause of the diminished α isozyme activity, whereas NAD+/NADH redox state imbalances appear to result in increased β isozyme activity. To support these ideas, treatment with ARI ameliorated the reduced PKCα activity in rat Schwannoma cells [42] and the enhanced PKCβ activity in endothelial and smooth muscle cells [59,60]. However, since the clinical trials of PKCβ inhibitors toward patients with DPN resulted in failure, research into PKC related to DPN, including its cross-talk with the AR/polyol pathway, has been on the decline.

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DG and MG exhibit direct toxicity toward Schwann cells [33,53,54]. Recent studies suggest the formation of AGEs from exogenous (diet-derived) and endogenous (polyol-pathway-derived) fructose, and the fructose-derived AGEs have been attracting attention as a novel pathogenic factor for a variety of metabolic, inflammatory, and neurodegenerative diseases [55,56].

2.2.4. PKC Activity Abnormalities Hyperglycemic insults have been shown to affect PKC activity in a cell- and isozyme- specific manner; PKCα activity is reduced in Schwann cells, whereas PKCβ activity is increased in vascular endothelial and smooth muscle cells [2,57]. These findings led us to suppose the involvement of α isozyme inactivation in DPN and β isozyme activation in microangiopathy (including reduced nerve blood flow), respectively. AR/polyol pathway is suggested to interact with PKC signaling cascades and differentially influence the expression/activity of the α and β isozymes [48,58]. As described above, myo-inositol depletion can be a cause of the diminished α isozyme activity, whereas NAD+/NADH redox state imbalances appear to result in increased β isozyme activity. To support these ideas, treatment with ARI ameliorated the reduced PKCα activity in rat Schwannoma cells [42] and the enhanced PKCβ activity in endothelial and smooth muscle cells [59,60]. However, since the clinical trials of PKCβ inhibitors toward patients with DPN resulted in failure, research into PKC related to DPN, including its cross-talk with the AR/polyol pathway, has been on the decline.

2.2.5. Epalrestat as a Pathogenesis-Based Medicine for DPN The accumulating evidence indicates that AR and the polyol pathway are major cul- prits in the development and progression of DPN, and epalrestat, one of the AR inhibitors, has been developed and approved as a pathogenesis-based medicine in Japan (Table1). However, its efficacy has been limited to the patients with mild DPN and stable glycemic control [6], and such unsatisfactory outcomes may result from multiple pathogenic factors for DPN, such as the collateral glycolysis pathways other than the polyol pathway [2,4,9,12], vascular abnormalities [61], and lifestyle-related factors other than hyperglycemia (e.g., obesity, dyslipidemia, hypertension, and smoking) [62]. The detailed understanding of the pathological roles of AR in DPN, particularly its relevance to other factors, may be required for developing more effective therapies. Immortalized Schwann cells established from normal and AR-deficient mice will be beneficial tools for the therapeutic approaches toward DPN, as discussed in the following sections.

3. IMS32 Schwann Cells as a Useful Tool to Study AR and the Polyol Pathway under Diabetic Conditions 3.1. IMS32 Cells Have Been Utilized for the Study of DPN The culture system of Schwann cells is useful to investigate the high-glucose-induced alterations of mRNA/protein expression proﬁles and related metabolic pathways, including AR and the polyol pathway. Although the primary culture of rodent Schwann cells has been utilized for the study of DPN [63], a time-consuming process is needed to acquire good yields of Schwann cells with high purity. To avoid such an inconvenience, a variety of cell lines were established from Schwannoma cells and the long-term primary cultured Schwann cells by oncogene transfection or spontaneous immortalization [64]. Because of the high proliferative activity and uniformity, these cell lines are more suitable for molecular, biochemical, and metabolomic analyses than primary cultured Schwann cells. Conversely, the proliferative activity of the cell lines seems to conﬂict with the degree of differentiation and retention of Schwann cell property. A spontaneously immortalized mouse Schwann cell line IMS32 established from adult ICR mice [14] possesses both moderate growth potency and distinct Schwann cell phenotypes (e.g., spindle-shaped morphology, expression of glial cell markers, synthesis and secretion of neurotrophic factors and neuroprotective cytokines (Figure3)). In addition, the IMS32 cell culture does not require any growth stimulants, necessary for the primary culture of Schwann cells (e.g., neuregulin-β and Int. J. Mol. Sci. 2021, 22, 1031 6 of 14

forskolin) [57]. Because of these advantages, IMS32 cells have been utilized for exploring the pathogenesis of DPN [12,15,33,65–73] (Table2) and amyloid polyneuropathy [ 74], as well as the mechanisms of action of axonal regeneration-promoting molecules (e.g., ciliary neurotrophic factor [75], sonic hedgehog [76], and oxidized galectin-1 [77]). IMS32 cells are one of the best characterized Schwann cell lines and currently available for purchase at several companies. In contrast to these Schwann cell-like phenotypes, IMS32 cells differ from primary cultured Schwann cells in that the former are not contact-inhibited and that they form ball-shaped subcolonies when cultures reach conﬂuence [14]. In addition, we have not succeeded in demonstrating that IMS32 cells myelinate neurites in co-culture with neurons. The higher proliferative activity of IMS32 cells compared with primary cultured Schwann cells might impede continuous and stable neuron-Schwann cell interplay, which usually take a month or longer to form the myelin structure. In mature peripheral nerves, Schwann cells play a key role in the conduction of nerve impulses, trophic support for neurons, production of extracellular matrix, regulation of immune responses, and axonal re- Int. J. Mol. Sci. 2021generation, 22, x FOR PEER after REVIEW injury [8,29]. Based on the ﬁndings, as described above, IMS32 cells appear7 of 16

to mimic some but not all characteristic features of naïve and activated Schwann cells.

Figure 3. IMS32 cells showed distinct Schwann cell phenotypes such as spindle-shaped morphology under a phase-con- Figure 3. IMS32 cells showed distinct Schwann cell phenotypes such as spindle-shaped morphology trast microscope (a) and immunoreactivity to p75 low-affinity neurotrophin receptor (p75NTR) (b). Modified from Sango et al., Experimentalunder Diabetes a phase-contrast Research 374943 microscope (2011) [16] ((Thea) and publisher immunoreactivity grants us the right to p75to reuse low-affinity the figure neurotrophinin other publications). receptor (p75NTR)(b). Modified from Sango et al., Experimental Diabetes Research 374943 (2011) [16] (The publisher grants us the right to reuse the figure in other publications). Table 2. IMS32 Schwann cells utilized for the study of diabetic peripheral neuropathy (review articles are not included).

Table 2. IMS32References Schwann cells utilized for theMajor study Findings of diabetic peripheral neuropathy (review articles are not included). Sango et al. (2006) [15] High-glucose (≥30 mM) conditions increased AR mRNA/protein expression and the in- References Major Findings tracellular contents of sorbitol and fructose. High-glucose (≥30 mM) conditions increased AR mRNA/protein expression and the intracellular contents of Sango et al. (2006) [15Ota] et al. (2007) [65] Metformin inhibited MG-induced apoptosis via JNK signaling pathway. sorbitol and fructose. Ota et al. (2007) [65]Sango et al. Metformin(2008) [33] inhibited MG-inducedBoth high-glucose apoptosis and via MG-induced JNK signaling upregulation pathway. of AR and oxidative stress markers Both high-glucose and MG-induced upregulation of AR and oxidative stress markers (4-hydroxy-2-nonenal, Sango et al. (2008) [33] (4-hydroxy-2-nonenal, acrolein and hexanoyl lysine). Tosaki et al.acrolein (2008) [66] and hexanoyl Hyperglycemic lysine). insults inhibited nerve growth factor (NGF) secretion from IMS32 cells, Hyperglycemic insults inhibited nerve growth factor (NGF) secretion from IMS32 cells, being a cause of reduced Tosaki et al. (2008) [66] neurite outgrowth activitybeing of a thecause conditioned of reduced media. neurite outgrowth activity of the conditioned media. Kim et al. (2011)A mixture [67] extract of Dioscorea A mixture japonica extractThunb of Dioscorea and Dioscorea japonica nipponica ThunbMakino and exertedDioscorea neurite nipponica Makino exerted Kim et al. (2011) [67] outgrowth-promotingneurite activity outgrowth-promoting on dorsal root ganglion (DRG) activity neurons, on do butrsal not root NGF ganglion induction (D effectsRG) neurons, on primary but not cultured and IMS32 SchwannNGF induction cells. effects on primary cultured and IMS32 Schwann cells. Long-term (>8 wk) hyperglycemic insults up-regulated the expression of genes that promote glycolytic pathway Kim et al. (2013) [68]Kim et al. (2013)and down-regulated [68] theLong-term expression (> of8 geneswk) hyperglycemi involved in fattyc insults acid metabolism, up-regulated pentose–phosphate the expression pathway of genes and that TCA cycle. promote glycolytic pathway and down-regulated the expression of genes involved in Hyperglycemic insults induced Schwann cell de-differentiation and suppressed insulin-like growth factor 1 Hao et al. (2015) [69] fatty acid metabolism, pentose–phosphate pathway and TCA cycle. expression via polyol pathway hyperactivity. Hao et al. (2015)Hyperglycemic [69] insults Hyperglycemic enhanced AR expression, insults induced lipid peroxidation, Schwann cell and de-differentiation caspase-3 activity in and a time-dependent suppressed insulin- Cinci et al. (2015) [70] manner (2 days < 7 dayslike< growth 14 days). factor 1 expression via polyol pathway hyperactivity. Human mobilized mononuclear cells (hMNC) restored DPN in STZ-mice and enhanced the expression of myelin Min et al. (2018) [71]Cinci et al. (2015) [70] Hyperglycemic insults enhanced AR expression, lipid peroxidation, and caspase-3 ac- protein zero in co-culturedtivity IMS32in a time-dependent cells through hepatocyte manner growth (2 days factor-paracrine < 7 days < 14 activity.days). Omega-3 polyunsaturated fatty acids alleviated oxidative stress-induced cell death by activating the antioxidant Tatsumi et al. (2019) [72] Min et al. (2018)enzymes [71] through the Human Nrf2 pathway. mobilized mononuclear cells (hMNC) restored DPN in STZ-mice and enhanced Recurrent short-termthe hypoglycemic expression (2.5 of mM)myelin and protein hyperglycemic zero in (25 co-cultured mM) insults IMS32 induced cells apoptosis through and hepatocyte Kato et al. (2019) [73] oxidative stress via thegrowth ER stress factor-paracrine response. activity. Mizukami et al. (2020) [12] Glucosamine induced IMS32 cell death via insulin signaling impairment and ATP depletion. Tatsumi et al. (2019) [72] Omega-3 polyunsaturated fatty acids alleviated oxidative stress-induced cell death by activating the antioxidant enzymes through the Nrf2 pathway. Kato et al. (2019) [73] Recurrent short-term hypoglycemic (2.5 mM) and hyperglycemic (25 mM) insults induced apoptosis and oxidative stress via the ER stress response. Mizukami et al. (2020) [12] Glucosamine induced IMS32 cell death via insulin signaling impairment and ATP depletion.

3.2. IMS32 Cells are Suitable for Exploring AR/Polyol Pathway-Related Abnormalities in DPN In primary cultured Schwann cells [28] and a Schwannoma-derived cell line JS1 [27], AR expression and the polyol contents were unaltered in response to the hyperglycemic (20–30 mM) insults unless hyperosmotic stress (≥ 100 mM) was applied. In contrast, under exposure to high glucose (≥ 30 mM) conditions, we observed significant increases in AR mRNA/protein expression and the contents of sorbitol and fructose in IMS32 cells. Fur- ther, application of an AR inhibitor fidarestat (SNK-860, provided by Sanwa Kagaku Kenkyusho, Inabe, Japan) (Table 1) to the hyperglycemic milieu significantly diminished

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3.2. IMS32 Cells Are Suitable for Exploring AR/Polyol Pathway-Related Abnormalities in DPN

Int. J. Mol. Sci. 2021, 22, x FOR PEER REVIEWIn primary cultured Schwann cells [28] and a Schwannoma-derived cell line JS1 [278 ],of 16 AR expression and the polyol contents were unaltered in response to the hyperglycemic (20–30 mM) insults unless hyperosmotic stress (≥100 mM) was applied. In contrast, under exposure to high glucose (≥30 mM) conditions, we observed significant increases in AR themRNA/protein polyol contents expression [15,33]. and Inthe agreement contentsof with sorbitol our andstudy, fructose Cint ini et IMS32 al. (2015) cells. Further,[70] reported ap- theplication high glucose-induced of an AR inhibitor fidarestatupregulation (SNK-860, of AR provided activity by and Sanwa expression Kagaku accompanied Kenkyusho, Inabe, by en- hancedJapan) (Tablelipid1 peroxidation) to the hyperglycemic and caspase milieu significantly3 activity in diminished IMS32 cells. the polyolThese contents findings [ 15 suggest,33]. thatIn agreement the culture with of IMS32 our study, cells Cintiunder et high-glucose al. (2015) [70] conditions reported the can high be glucose-induceda useful in vitro model up- toregulation study AR/polyol of AR activity pathway-related and expression abnormalities accompanied in by DPN. enhanced Why lipid the peroxidationglucose concentra- and tionscaspase corresponding 3 activity in IMS32 to the cells. plasma These level findings in poorly suggest controlled that the culturediabetic of patients IMS32 cells (20–30 under mM) acceleratehigh-glucose the conditions polyol pathway can be a in useful IMS32,in vitro butmodel not in to primary study AR/polyol cultured pathway-related Schwann cells or otherabnormalities Schwann in cell DPN. lines, Why remains the glucose elusive. concentrations Sorbitol can corresponding be released to from the plasma IMS32 level cells in into poorly controlled diabetic patients (20–30 mM) accelerate the polyol pathway in IMS32, but the culture media (Sango et al., unpublished data), but the cells might possess a much not in primary cultured Schwann cells or other Schwann cell lines, remains elusive. Sorbitol greater capacity than other Schwann cells to store sorbitol and other glucose-derived me- can be released from IMS32 cells into the culture media (Sango et al., unpublished data), but tabolites.the cells might possess a much greater capacity than other Schwann cells to store sorbitol and otherThe glucose-derived expression of metabolites. AR in IMS32 cells was upregulated by exposure to MG (0.5 mM) underThe normoglycemic expression of (5.6 AR mM) in IMS32 conditions, cells was as upregulated well as exposure by exposure to hyperglycemic to MG (0.5 (30 mM) mM) insultsunder normoglycemic[33], as expected, (5.6 since mM) conditions,AR is known aswell to be as involved exposure toin hyperglycemicthe reduction (30of mMMG) (cf. Sectioninsults 2.1.2). [33], as However, expected, the since cellular AR is reactions known to to be MG involved were different in the reduction from those of to MG the (cf. high glucose.Section The2.1.2 MG-induced). However, thedose-dependent cellular reactions cell death to MG was were not different associated from with those the to increase the inhigh sorbitol glucose. and Thefructose MG-induced contents. dose-dependent On the other hand, cell death the high was notglucose associated insults with led theto the polyolincrease pathway in sorbitol hyperactivity and fructose without contents. a sign On theificant other influence hand, the on high the glucosecell viability insults led (Figureto the polyol4). These pathway findings hyperactivity suggest that without MG-induced a significant upregulation influence of on AR the under cell viability normoglycemic(Figure conditions4 ). These is findings a consequence suggest of that cytoprot MG-inducedective reactions, upregulation and of the AR amount under normo-of glucose availableglycemic conditionsfor utilization is a consequence through the of polyol cytoprotective pathway reactions, appears and to thebe amountinsufficient of glucose to cause available for utilization through the polyol pathway appears to be insufficient to cause sorbitol accumulation. However, to prevent IMS32 cell death, the efficacy of AR for MG sorbitol accumulation. However, to prevent IMS32 cell death, the efficacy of AR for MG detoxification is insufficient. detoxification is insufficient.

Figure 4. (a) High-glucose- and MG-induced upregulation of AR by Western blot analysis. (b) Intracellular contents of Figure 4. (a) High-glucose- and MG-induced upregulation of AR by Western blot analysis. (b) Intracellular contents of sorbitol (upper panel) and fructose (lower panel) after 7 days of exposure to (Glc-5.6mM), (Glc-30mM), and (MG-0.5mM) in sorbitol (upper panel) and fructose (lower panel) after 7 days of exposure to (Glc-5.6mM), (Glc-30mM), and (MG-0.5mM) µ p in thethe presencepresence or or absence absence of of SNK-860 SNK-860 (1 (1M). μM). Values Values represent represent the mean the mean + SEM + ofSEM 6 experiments. of 6 experiments. * < 0.01 * p as < compared0.01 as compared with with(Glc-5.6mM) (Glc-5.6mM) and and # p <# 0.01p < 0.01 as compared as compared with with (Glc-30mM). (Glc-30m ModiﬁedM). Modified from Sangofrom Sango et al., Openet al., Diabetes Open Diabetes J. 2008, J.1, 2008 1–11, [133, 1–11] [33](The (The publisher publisher grants grants us theus rightthe right to reuse to reuse the ﬁgure the figure in other in other publications). publications).

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According to the study by Hao et al. (2015) [69], primary cultured and IMS32 Schwann cells de-differentiated into immature cells (i.e., reduced expression of myelin proteins and enhanced expression of p75NTR, an immature Schwann cell marker) under hyperglycemic conditions via sorbitol accumulation. Treatment with epalrestat, an existing ARI used in Japan (Table1)[ 6,78], ameliorated these changes. That study suggests the involvement of the polyol pathway in Schwann cell de-differentiation, which may be a cause of demyelination under diabetic conditions. However, ultrastructural studies of the peripheral nerves from patients with DPN at early stages showed axonal degeneration of myelinated and unmyelinated fibers without overt demyelination [79]. In addition, AR transgenic mice exhibited more severe neurological manifestations and nerve fiber atrophy than non- transgenic littermates when they were rendered diabetes by STZ, but no mice developed demyelination under that condition [80]. These findings suggest that the main pathology of DPN is axonal degeneration, which precedes demyelination observed in patients with DPN at advanced stages. Because axon−Schwann cell interplay is essential for the maintenance of peripheral nerve function, its discordance due to Schwann cell de-differentiation might affect both myelinated and unmyelinated fibers. By employing IMS32 cells, we observed that novel ARIs such as fidarestat and ranirestat (AS-3201, provided by Sumitomo Dainippon Pharma Co., Ltd., Osaka, Japan) (Table1 ) suppressed the high-glucose-induced upregulation of sorbitol and fructose contents more potently than epalrestat (Sango et al., unpublished data). Galactose is recognized the pre- ferred substrate for AR, rather than glucose, and AR catalyzes the conversion of galactose to galactitol [81]. In our preliminary study, ranirestat suppressed galactose (25 mM)- induced upregulation of galactitol contents in IMS32 cells more effectively than epalrestat (Yako et al., in preparation). Despite the potent inhibitory effects against AR described above and the potential efficacy for patients with DPN, neither fidarestat nor ranirestat showed detectable benefits through the clinical trials [82].

4. Establishment of an AR-Deficient Schwann Cell Line IKARS1 4.1. Establishment and Characterization of IKARS1 Cells Spontaneously immortalized Schwann cells were established not only from normal mice, but from murine disease models (e.g., Charcot–Marie-Tooth disease, neurofibro- matosis and lysosomal storage diseases) [83]. These cell lines sufficiently represent the pathological features of the mutant mice, and they will be beneficial tools for studying the PNS lesions in the relevant diseases. In a similar manner, we obtained immortalized Schwann cell lines from AR-knockout C57BL/6 mice. Because our first trial to establish Schwann cell lines from wild-type littermates ended in failure, we employed one of the cell lines established from normal C57BL/6 mice in our institute as control of the AR- knockout cell line. These cell lines, termed IKARS1 (immortalized knockout AR Schwann cells 1) and 1970C3, respectively, showed distinct Schwann cell phenotypes similar to IMS32 cells, such as spindle-shaped morphology, expression of glial cell markers (S100β, p75 low-affinity neurotrophin receptor, etc.) and synthesis/secretion of neurotrophic factors [13]. Conditioned media collected from IKARS1 and 1970C3 cells enhanced neurite outgrowth from cultured adult mouse DRG neurons, and both cell lines secreted NGF and glial-cell-line-derived neurotrophic factor (GDNF) into the culture medium. The deficient AR activity in IKARS1 cells was confirmed by the galactose (25 mM)-induced increase in galactitol contents in 1970C3 cells but not in IKARS1 cells. A marked down-regulation of mRNA/protein expression of the enzymes downstream of AR in the polyol pathway (i.e., SDH and ketohexokinase (KHK)) in IKARS1 cells compared with 1970C3 cells (Figure5) is consistent with the high-glucose-(30 mM)-induced increases in the sorbitol and fructose contents in 1970C3 cells but not IKARS1 cells. These findings indicate the absence of the polyol pathway in IKARS1 cells. It is also noteworthy that mRNA expression of aldo-keto reductases (AKR1B7 and AKR1B8) and aldehyde dehydrogenases (ALDH1L2, ALDH5A1, and ALDH7A1) was significantly up-regulated in IKARS1 cells compared with 1970C3 cells (Figure5). AKR1B7 Int. J. Mol. Sci. 2021, 22, 1031 9 of 14

and AKR1B8 are considered AR-related enzymes because of the high degree of sequence similarities to AR [52], and the AKRs and ALDHs that are up-regulated in IKARS1 cells appear to be involved in the reduction and oxidation of various aldehydes [84]. Although Int. J. Mol. Sci. 2021, 22, x FOR PEER REVIEWAR is suggested to be an efficient catalyst for the reduction in 4HNE, MG, and10 3-DG of 16 (cf. Section 2.1.2), the cell viability assays revealed no significant differences in the relative survival ratios between IKARS1 and 1970C3 cells after exposure to these substances. The findings that AR gene deletion does not augment the aldehyde toxicity imply functional redundanciesredundancies among among AKRs AKRs and and ALDHs ALDHs regarding regarding the the metabolic metabolic disposal disposal system system of ofthese these aldehydesaldehydes in inSchwann Schwann cells. cells. Because Because exposure exposure to tothese these aldehydes aldehydes further further up-regulated up-regulated mRNAmRNA expression expression of ofAKR1B7 AKR1B7 and and AKR1B8, AKR1B8, but but not not ALDHs, ALDHs, in inIKARS1 IKARS1 cells, cells, the the reactive reactive aldehydealdehyde detoxification detoxification can can be be taken taken over over by by AKR1B7 AKR1B7 and and AKR1B8 AKR1B8 in inthe the absence absence of ofAR. AR.

Figure 5. Establishment and characterization of IKARS1 cells. Marked down-regulation of polyol Figurepathway-related 5. Establishment enzymes and (SDH,characterization KHK) mRNA of IKARS1 expression, cells. and Marked significant down-regulation upregulation of of polyol aldo-keto pathway-relatedreductases (AKR1B7, enzymes AKR1B8) (SDH, KHK) and aldehyde mRNA expres dehydrogenasession, and significant (ALDH1L2, upregulation ALDH5A1, of ALDH7A1) aldo- ketomRNA reductases expression (AKR1B7, in IKARS1 AKR1B8) cells and compared aldehyde with dehydrogenases those in 1970C3 (ALDH1L2, cells. The upregulatedALDH5A1, enzymes ALDH7A1)might take mRNA over the expression AR detoxifying in IKARS1 function. cells The comp genesared encoding with those the respectivein 1970C3 enzymescells. The are upregu- expressed latedas theirenzymes abbreviations might take in over square the brackets. AR detoxifying function. The genes encoding the respective enzymes are expressed as their abbreviations in square brackets. 4.2. Establishment of IWARS1 Cells and Future Studies with IKARS1 and IWARS1 4.2. EstablishmentThe second of trialIWARS1 to establish Cells and Schwann Future Studies cell lines with fromIKARS1 wild-type and IWARS1 littermates of AR- deﬁcientThe second C57BL/6 trial mice to establish was successful. Schwann One cell of thelines cell from lines wild-type termed IWARS1 littermates (immortalized of AR- deficientwild-type C57BL/6 AR Schwann mice was cells successful. 1) possess One distinct of the Schwann cell lines cell termed phenotypes IWARS1 similar (immortal- to 1970C3 izedcells wild-type (Niimi etAR al., Schwann in preparation). cells 1) possess Unlike distinct IMS32 cells,Schwann neuregulin- cell phenotypesβ is required similar for to the 1970C3passage cells of (Niimi IKARS1, et al., 1970C3 in preparation). and IWARS1 Unlike cells. IMS32 The lower cells, growthneuregulin- potencyβ is required of these for cells thecompared passage of with IKARS1, IMS32 1970C3 cells might and IWARS1 be advantageous cells. The for lower myelination growth potency under co-culture of these cells with comparedneurons, with although IMS32 we cells have might not yetbe advantageous conﬁrmed that for they myelination possess this under capability. co-culture with neurons,The although IWARS1 we cells have are not “true yet confirmed controls” ofthat IKARS1 they possess cells, this and capability. these cell lines can be utilizedThe IWARS1 to explore cells various are “true collateral controls” glucose-utilizing of IKARS1 cells, pathways and these (polyolcell lines pathway, can be uti- hex- lizedosamine to explore pathway, various PKC collateral pathway andglucose-utilizing AGE pathway) pathways in relation (polyol to AR pathway, (Figure6 ).hex- The osamineAR knockout pathway, mice, PKC when pathway rendered and AGE diabetes pathway) by streptozotocin, in relation to showedAR (Figure defense 6). The against AR neurological manifestations by 12 weeks after onset of diabetes [11]. However, Mizukami knockout mice, when rendered diabetes by streptozotocin, showed defense against neu- et al. (2020) [12] recently reported the reduced NCV in AR knockout mice with 16 weeks rological manifestations by 12 weeks after onset of diabetes [11]. However, Mizukami et of diabetes, suggesting that other pathways, downstream or independent of the polyol al. (2020) [12] recently reported the reduced NCV in AR knockout mice with 16 weeks of pathway, are involved in the pathogenesis of DPN with long duration of hyperglycemia. diabetes, suggesting that other pathways, downstream or independent of the polyol path- They also observed an accumulation of glucosamine in the sciatic nerves of diabetic AR way, are involved in the pathogenesis of DPN with long duration of hyperglycemia. They knockout and wild-type mice and direct toxicity of glucosamine toward cultured DRG also observed an accumulation of glucosamine in the sciatic nerves of diabetic AR knock- neurons and IMS32 Schwann cells. While how glucosamine is up-regulated under diabetic out and wild-type mice and direct toxicity of glucosamine toward cultured DRG neurons conditions remains unclear, glucosamine can be metabolized to glucosamine-6-phosphate, and IMS32 Schwann cells. While how glucosamine is up-regulated under diabetic condi- constituents of the hexosamine pathway, the second collateral glycolysis pathway [2,4]. tions remains unclear, glucosamine can be metabolized to glucosamine-6-phosphate, constituents of the hexosamine pathway, the second collateral glycolysis pathway [2,4]. IKARS1 and IWARS1 cells will be essential reagents to determine the involvement of the AR and hexosamine pathway in the pathogenesis of DPN, as well as other pathogenic factors of DPN related to the polyol pathway, such as glycated, oxidative and nitrosative stress (cf. Section 2.2).

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IKARS1 and IWARS1 cells will be essential reagents to determine the involvement of the Int. J. Mol. Sci. 2021, 22, x FOR PEER REVIEW 11 of 16 AR and hexosamine pathway in the pathogenesis of DPN, as well as other pathogenic factors of DPN related to the polyol pathway, such as glycated, oxidative and nitrosative stress (cf. Section 2.2).

Figure 6. IWARS1 and IKARS1 cells established from wild-type and AR-deﬁcient C57BL/6 mice as usefulFigure tools 6. IWARS1 for exploring and IKARS1 collateral cells glucose-utilizing established from pathways. wild-type Our and current AR-deficient investigation C57BL/6 using mice these as celluseful lines tools focuses for exploring on the mechanisms collateral glucose-ut of glucosamineilizing accumulation pathways. Our in thecurrent PNS investigation under hyperglycemic using conditionsthese cell lines regardless focuses of on the the presence mechanisms or absence of glucosamine of AR, and itsaccumulation association within the hexosamine PNS under pathway hyper- hyperactivityglycemic conditions that may regardless impair insulin of the signaling.presence or absence of AR, and its association with hexosamine pathway hyperactivity that may impair insulin signaling. 5. Conclusions 5. ConclusionsAs we stated in the title of this paper, the involvement of AR and the polyol pathway in SchwannAs we cell stated dysfunction in the title under of this diabetic paper, conditions the involvement and the pathogenesisof AR and the of polyol DPN pathway is an old andin Schwann new target cell for dysfunction developing under effective diabetic therapies conditions against and the the diabetic pathogenesis complications, of DPN suchis an asold DPN. and new Despite target growing for developing evidence, effective as illustrated therapies in Section against2, researchersthe diabetic studying complications, DPN facesuch the as realityDPN. Despite that much growing basic and evidence, clinical as research illustrated work in is Section still required. 2, researchers As described studying in SectionDPN face3, IMS32 the reality cells underthat much hyperglycemic basic and conditionsclinical research are a useful work isin still vitro required.model to As study de- thescribed polyol-pathway-related in Section 3, IMS32 abnormalities.cells under hype Therglycemic IKARS1 andconditions IWARS1 are cells a useful introduced in vitro in Sectionmodel to4 are study expected the polyol-pathway-related to shed some light on abnormalities. the cross-talk The between IKARS1 the and polyol IWARS1 pathway cells andintroduced other pathogenic in Section 4 factors are expected of DPN. to shed We look some forward light on to the the cross-talk exponential between knowledge the pol- progressionyol pathway in and the other novel pathogenic understanding factors of ARof DPN. and related We look pathways forward in to DPN the usingexponential these Schwannknowledge cell progression lines through in the international novel understanding collaborative of efforts. AR and related pathways in DPN using these Schwann cell lines through international collaborative efforts.

Author Contributions: N.N. and K.S. designed and wrote the manuscript. H.Y., S.T. and S.K.C. wrote the manuscript. All authors have read and agreed to the published version of the manuscript.

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Author Contributions: N.N. and K.S. designed and wrote the manuscript. H.Y., S.T. and S.K.C. wrote the manuscript. All authors have read and agreed to the published version of the manuscript. Funding: This work was supported in part by a Grant-in-Aid for Scientific Research from the Ministry of Education, Culture, Sports, Science, and Technology (KAKENHI 20K07773). Institutional Review Board Statement: Our study described in this article was conducted according to the guidelines of the Declaration of Helsinki, and approved by the Institutional Review Board of Tokyo Metropolitan Institute of Medical Science (institutional approval number 20-016, 2020). Informed Consent Statement: Not applicable. Data Availability Statement: Not applicable. Acknowledgments: We thank Koichi Kato and Hiroki Mizukami for helpful suggestions on our research described in this article. Conflicts of Interest: The authors declare no conflict of interest.

Abbreviations

AKR Aldo-keto reductases ALDH Aldehyde dehydrogenase AR Aldose reductase 3-DG 3-Deoxyglucosone DPN Diabetic peripheral neuropathy DRG Dorsal root ganglion GDNF Glial cell line-derived neurotrophic factor 4HNE 6-Hydroxy-2-nonenal IKARS1 Immortalized knockout aldose reductase Schwann cells 1 IMS32 Immortalized mouse Schwann cells 32 IWARS1 Immortalized wild-type aldose reductase Schwann cells 1 KHK Ketohexokinase MG Methylglyoxal NADPH Reduced nicotinamide adenosine dinucleotide phosphate NGF Nerve growth factor p75NTR p75 low-afﬁnity neurotrophin receptor PKC Protein kinase C PNS Peripheral nervous system SDH Sorbitol dehydrogenase

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