decreased glucoseuptakein muscleand adiposetissue,and dyslipidemia due to disturbances in the inhibition oflipolysis in adipose tissues (Chang-Chen etal,2008)(Fig1).
Fig.1.Pathogenesisoftype2diabetesmellitus.(StumvollM etal,2005)
2.Bet a-cel ldysf unct i on andf ai l ur edur i ng t ype2di abet es.
During the pastseveraldecades,pancreatic beta-celldysfunction has beenthoughttoplay an importantroleinthedevelopmentoftype2diabetes (DeFronzo,2009).Itisnow acceptedthat,wheninsulinresistancedevelopsin accordance with environmentalfactors such as obesity,individuals who are genetically susceptibletodiabetesfailtocompensatefortheincreasedinsulin demand such thatbeta-cellfailure finally occurs (Prentkiand Nolan,2006). Pancreaticbeta-cellsareadaptedtowardinsulinresistanceinaccordancewith increasing cellmass and activity during the early stage ofdiabetes.During early stage of insulin resistance, beta-cells secrete sufficient insulin to maintainnormalbloodglucoselevels.However,longterm insulinresistanceis characterized by insufficientinsulin secretion thatleads to impaired glucose tolerance.Ifthisstatepersists,impairments in thecontrolofglucoselevels, due to beta-cell dysfunction and death, become increasingly severe and eventually lead to diabetes.A quantitative reduction in beta-cellmass was revealedby insulin staining in isolated isletsoftype2diabetespatients,due mostly to increased apoptosis (Fig 2).Therefore,it is also important to elucidate the mechanisms underlying beta-celldysfunction and apoptosis,as wellasinsulinresistance.
Fig.2.Naturalhistory oftype2diabetesand beta-cellmass.(Sakuraba H etal,2002)
B.Cel l ul armechani smsunder l yi ng bet a-cel ll i pot oxi ci t y
Ithas been reported previously thathigh levels ofglucose or fatty acidsmay both bedetrimentaltobeta-cellfunction,butin combination these compoundsareeven moreharmful.Toxicity dueto freefatty acids (FFAs), which isknown aslipotoxicity and occursin obesetype2diabetespatients, probablymediatesbeta-cellfailure(Leonardietal,2003).Chronicexposureto high concentrations of fatty acids is related to the inhibition of glucose-stimulated insulin secretion and decreased ofinsulin geneexpression.
Furthermore,it has been demonstrated clearly thatlong-term exposure to fatty acids can induce beta-cellapoptosisin cultured cells orisolated islets;
palmitic and stearic fatty acids are particularly cytotoxic to beta-cells (Maedler et al, 2001; Maedler et al, 2003). However, the mechanism underlying fatty acid-induced beta-cell toxicity has not yet been clearly defined. Typically, FFAs are non-toxic to beta-cells when they are completely oxidized or synthesized to triglyceride.Incomplete oxidation of fatty acids,oraccumulation oflong-chain acyl-coenzyme A (LC-CoA)and lipid intermediatemolecules (such as lysophosphatidic acid,phosphatidicacid and diacylglycerol; DAG), may play an important role in FFA-induced toxicity (Prentki et al,2002).Ceramide and DAG can activate c-Jun N terminalkinase(JNK)andproteinkinaseC signaling;thesesignalsreportedly induce lipotoxicity by inhibiting insulin signaling (Shimabukuro etal,1998;
Eiteletal,2003)(Fig.3).
Fig.3.Lipid-induced insulin resistance.(KiensB,2006;EugeneDT and DanielGD,2012)
Endoplasmic reticulum (ER) stress is a critical mediator of
2012).Furthermore,JNK-dependentserinephosphorylation ofinsulin receptor substrate-1 (IRS-1),an inhibitory form ofIRS-1,links ER stress with the inductionofobesity-inducedinsulinresistance(Hotamisligil,2008).Ithasbeen suggested thatinsufficientactivation ofthe insulin signaling pathway also contributes to beta-celldamage (Heninge,2003).Although itremains to be determined how FFA induces the ER stress responses,FFA surplus may facilitate UPRs through ER Ca2+ depletion (Cunha etal,2008;Hara etal, 2014;Mandlet al,2009).Impaired ER-Golgitrafficking may also explain palmitate(PA)-inducedER stress.
Fig.4.Endoplasmicreticulum stresssignaling.(Cunhaetal,2008)
Collectively,lipotoxicity-induced beta-celldeath is mainly caused by apoptotic cell death. Although it is not yet fully understood how high concentrationsoffatty acidsinducemitochondrialdamage,itisbelieved that the activation of various stress signals plays a major role in mitochondria-mediated cell death. The release of cytochrome C from mitochondriaactivatescaspase9/caspase3,which isknown tobeaprimary mechanism underlying mitochondria-mediated cell death.In particular,our group hasdemonstrated thatoxidativeand ER stress,which resultsin JNK activation,plays a key role in lipotoxicity.Furthermore,activation of the NF-kB signaling pathway in turn activates inflammatory signals,which are reportedly involved in beta celldeath;because insulin signaling is reduced during lipotoxicity, decreased activity in the AKT signaling pathway is believedtoplaythemajorroleinfattyacid-inducedtoxicity(Fig.5).
Fig.5.
Cel l ul armechani sm under l yi ng bet a-cel ll i pot oxi ci t y.
C.Mi t ochondr i aldysf unct i on dur i ng l i pot oxi ci t y
humans(Bousheletal,2007;Hancocketal,2008).Furthermore,mitochondrial flavoprotein apoptosis-inducing factor knockout mice exhibit impaired mitochondrialOXPHOS activity butalso demonstrate increased resistance to obesity and diabetes induced by HFD (Pospisilik etal,2007);this suggests thattherelationship between mitochondrialfunction and insulin sensitivity iscomplex.
occurs when ATP and NADH levels are high,glycolysis is suppressed.
between 0.7 and 1.0 undernormalconditions,and providemitochondrialfuel. A highRQ indicatesarelatively highdegreeofglucoseoxidation,whereasa low RQ predominately reflectsfatoxidation (Fig.6).However,fluctuationsin RQ are blunted under insulin resistance conditions. Excess supply and increasedoxidationoffatty acidscanleadtotheaccumulationofacetyl-CoA, which allosterically activates pyruvate dehydrogenase kinase (PDK), and results in the inhibition ofPDH (Sugden and Holness,2006).In a mouse model, genetic inhibition of PDH in muscle and heart led to muscle hypertrophy and heart dysfunction after feeding with a HFD.In contrast, geneticupregulationofPDK orupregulationundercertainconditionsincluding HFD,fasting,oralackofinsulin,maintainsglucoseoxidationatalow level. This circumstance mimics a state of metabolic inflexibility and is characteristic ofinsulin resistance (Kelley and Mandarino,2000).Conversely, thefasting blood glucoselevelofPDK4-deficientmiceissignificantly lower compared with wild-type mice (Jeoung and Harris 2008),probably due to active PDH dividing pyruvate, a substrate of gluconeogenesis, into acetyl-CoA.TheseobservationsemphasizetheimportanceofPDH in glucose and lipid homeostasis.Furthermore,an increase in cellular citrate inhibits PFK-1,leading to reduced glycolysis,pyruvateoxidation and glucoseuptake (Fig.7).Although this metabolic inflexibility is recognized as a hallmark of heartmetabolicdisordersandisapotentialcauseofcellulardysfunction,itis stillnotfullyunderstoodatthemolecularlevel.
Fig.6.Metabolicinflexibility.(MuoioDM,2014)
Fig.7.Inhibition ofglucose oxidation by fatty acid utilization.(An D andRodriguesB,2006)
D.Rel at i onshi pbet ween i r on anddi abet es
(Rouault, 2006). Excessive iron separates IRP from IREs on the 3' untranslated region (UTR)oftransferrin mRNA and the 5'UTR offerritin mRNA.Therefore,the stability ofTfR mRNA is decreased,and additional iron uptake reduced,whereas the translation of ferritin is increased and sequestersironinsidethecell.Conversely,in casesofinsufficientiron within the cell,IRPs bind to the IREs ofthe 3'UTR in TfR mRNA,and the 5' UTR in ferritin mRNA,resulting in highly stable TfR mRNA and ferritin translation inhibition,which ultmately increases iron contentwithin the cell (Tong andRouault,2007;Simcox andMcClain,2013;Gabrielsen etal, 2012) (Fig.9).
Fig.8.Iron metabolism.(RouaultTA andTongWH,2005)
Fig.9.Regulation oftransferrin receptorand ferritin by iron regulatory protein.(CazzolaM andSkodaRC,2000)
2.Rol eofi r on i n met abol i sm
3.I r on and met abol i csyndr ome
Ironhomeostasisiscriticalformaintainingnormalcellfunctionbecause iron playsan importantrolein mitochondrialrespiratory metabolism.In fact, iron acts as an electron transfer component during oxidation/reduction reactions thatoccur in cytochrome,severalmitochondrialelectron transport complex (iron-sulfurcluster)and theTCA cycle(Leviand Rovida,2009;Ye and Rouault,2010).Itwasthoughtpreviously thatiron overload may cause insulin resistance. Peripheral insulin resistance is increased, and insulin secretion decreased,when body iron levels rise.Conversely,the removalof ironrestoresinsulinsensitivity andsecretion(Abraham etal,2006).Although excessiveiron,asindicatedbyhyperferritinemiaandliverirondeposition,isa typicalfeatureofmetabolicsyndrome,severeobesity isusually accompanied by a lack ofiron.Theassociation between obesity and low iron levels has been evaluated previously (Ku etal,2009).Ithas been reported thatiron deficiencycausescelldeathandgrowtharrest,whereasexcessirongenerates free radicals that also structurally and functionally damage cellular biomolecules such as DNA,membrane lipids and proteins and,ultimately, inducescelldeath(Andersonetal,2012;Muckenthaleretal,2008).Therefore, cellularironlevelsmustbetightlycontrolled.
D.Sodi um f l uor oacet at e
Fig.10.Synthesisoffluorocitrate.(LaubleH etal,1996)
Fig.11.Synthesisof4-hydroxy-trans-aconitateby aconitase.(LaubleH etal,1996)
2.Ef f ectofSFA on gl ucosemet abol i sm
E.Obj ect i ve
Chronic exposure to high levels offatty acids can induce beta-cell death.However,the mechanism underlying this celldeath is notcompletely understood.Iron acts as a cofactor in the TCA cycle,and for electron transferchain enzymes.In the presentstudy,Ifound thatsupplementation with iron reduced PA-induced INS-1 celldeath.Therefore,Iattempted to determine the mechanism underlying the protective effects of iron against PA-induced lipotoxicity.Furthermore,Ialso found thatsodium fluorocitrate (SFC),which is a known aconitase inhibitor,completely protected against PA-induced INS-1 celldeath.Ifthe mechanism by which SFC completely protectsagainstPA-induced celldeath could beclarified,itmay bepossible to prevent lipotoxicity. Therefore, I also attempted to determine the mechanism underlying the protective effects of SFC against PA-induced lipotoxicity.Aco1fulfillstwofunctions,actingbothasanenzymeandanIRP.
Thus,experimentswerealsoconducted to determinetheassociation between iron metabolism and lipotoxicity,assuming thataconitase inhibited by SFC mayplayaroleasanIRP.