this process requires insulin , a peptide hormone that lowers glycemia by triggering the uptake of glucose into cells . as the exclusive source of insulin , pancreatic cells play an essential role in glucose homeostasis .
they are responsible for the production , storage and release of insulin in a manner that is strictly coupled to demand .
what is clear is that glucosestimulated insulin secretion ( gsis ) is a complex and tightly regulated process , the disruption of which has severe consequences .
evidence of this can be seen in diabetes , a widespread metabolic disease resulting from the failure of cells to meet the physiological requirements for insulin . in type  1 diabetes , this is caused by the autoimmunemediated destruction of cells . in type  2
diabetes , cells fail to adequately compensate for the insulin resistance often ensuing in conditions such as obesity . early cell dysfunction , characterized by impaired gsis , is considered fundamental in this process .
the deficits in gsis observed in type  2 diabetes have been attributed to numerous factors .
cholesterol is an essential constituent of mammalian cell membranes , which influences their fluidity , permeability and curvature .
it is also a major component of membrane rafts  dynamic nanoscale assemblies of sterols , sphingolipids and proteins that can be stabilized to coalesce into platforms that facilitate various cellular processes .
these domains are now widely considered to participate in the regulation of gsis , a conclusion based primarily on two observations .
first , numerous proteins involved in either gsis or the biogenesis of secretory granules ( sgs ) , the intracellular storage organelles from which insulin is secreted , have been shown to partition with detergentresistant membranes ( drms ) in the light fractions of sucrose density gradients after solubilization at 4c with triton x100 or other nonionic detergents .
second , perturbation of membrane cholesterol often induced a repartitioning of these proteins that corresponded with alterations in their function , as well as in insulin secretion .
these results have prompted speculation that changes in membrane rafts , triggered by altered cellular cholesterol homeostasis , contribute to the impaired gsis in type  2 diabetes .
here we provide a brief overview of the work linking membrane rafts and gsis , and the insight it provides into cell dysfunction .
recent studies in mice have emphasized the importance of cell cholesterol homeostasis for insulin secretion .
disruption of cholesterol efflux through knockout of the cholesterol transporter adenosine triphosphate ( atp)binding cassette transporter  a1 ( abca1 ) selectively in cells resulted in the accumulation of islet cholesterol , as well as glucose intolerance and impaired gsis .
this was in agreement with the previous observation that knockout of liver x receptor  ( lxr ) , a nuclear hormone receptor that upregulates abca1 expression in response to cholesterol , caused cell dysfunction .
mice lacking the cholesterol acceptor apolipoprotein  e ( apoe ) also showed elevated islet cholesterol levels and reduced insulin secretion . unlike abca1 mice , however , circulating cholesterol levels in apoe mice were elevated , suggesting a correlation between hypercholesterolemia and cell dysfunction .
knockout of the low density lipoprotein receptor ( ldlr ) , which facilitates cholesterol uptake , also triggered hypercholesterolemia , although its impact on cell function was inconsistent .
one group observed elevated islet cholesterol levels , impaired glucose tolerance and reduced gsis in these mice , whereas another reported no significant alterations in islet cholesterol or cell function . despite this discrepancy , the trend emerging from these and other studies is that cholesterol accumulation in cells impairs insulin secretion .
work investigating the role of membrane rafts in gsis has identified numerous stages at which this might occur .
glycemia is normally maintained at concentrations of 5  mmol / l , and increases above this value trigger glucose uptake in cells . in rodents ,
this is facilitated primarily by glucose transporter ( glut)2 , whereas in humans glut1 is the predominant glucose transporter .
although there is little direct evidence for the association of these transporters with membrane rafts in cells , glut1 partitioned with drms in various other cell types .
in addition , cholesterol depletion of liverderived clone  9 cells with methylcyclodextrin ( mcd ) , which extracts cholesterol from the plasma membrane , disrupted the raft partitioning of glut1 and enhanced glucose transport .
interestingly , ldlr islets with elevated cholesterol showed reduced glucose uptake , as did cholesterolloaded primary cells .
however , the possibility that this represented a membrane raftdependent effect on glucose transporter activity was not investigated .
this critical ratelimiting step in glucose metabolism is catalyzed by glucokinase ( gck ) , the primary glucose sensor of cells .
a pool of gck associates with sgs , and glucose induces its dissociation and activation .
this association is mediated by an interaction with neuronal nitric oxide synthase ( nnos ) , and recent work suggests a role for membrane rafts in this process .
have reported that mcd reduced nnos dimerization , promoted the translocation and activation of gck , and enhanced gsis .
nnos has been previously shown to interact with islet cell autoantigen  512 ( ica512 ) , a transmembrane sg protein also known as insulinomaassociated protein  2 ( ia2 ) or protein tyrosine phosphatase , receptor type , n ( ptprn ) .
hao et  al . speculated that ica512 associates with membrane rafts , and this facilitates nnos dimerization and the retention of gck on sgs ( figure  1a ) .
both ica512 and nnos partition with drms in insulinoma cells ( dirkx r and solimena m , unpublished data , 20072009 ) , and recent structural studies have indicated that the luminal / extracellular domain of ica512 dimerizes .
however , it is not yet clear if membrane rafts influence ica512 dimerization or its interaction with nnos . therefore , additional work is required , especially in light of reports that gck does not translocate from granules in response to glucose and that nnitrolarginine methyl ester ( lname ) , an inhibitor of nnos that enhanced its dimerization , amplified insulin secretion .
apart from gck activity , cholesterol overloading of cells directly disrupted mitochondrial metabolism , and this together with reduced glucose uptake likely accounted for the near complete inability of glucose to elicit increases in cellular atp levels in these cells . 
this initiates ca influx , leading to soluble nethylmaleimidesensitive factor attachment protein receptor ( snare)mediated secretory granule ( sg ) fusion and insulin secretion .
sphingolipid and cholesterolenriched membrane rafts ( highlighted in red ) have been proposed to regulate various aspects of glucosestimulated insulin secretion including ( a ) glucose metabolism , and the secretiondependent translocation and activation of a sgassociated pool of glucokinase ( gck ) ; ( b ) katp , cav and kv channel activity , snaremediated sg fusion and its spatial coupling to ca entry , fusion pore formation , and the syntaxinmediated regulation of channel gating ; and ( c ) targeting and retention of prohormones , their processing enzymes , and granins in the cholesterolenriched membranes of newly forming sg .
cav , voltagegated ca ; cga , chromogranin a ; cpe , carboxypeptidase e ; glut , glucose transporter ; katp , adenosine triphosphatesensitive k ; kir , inwardly rectifying k ; kv , voltagegated k ; sur , sulphonylurea receptor ; nnos , neuronal nitric oxide synthase ; ia2 , insulinomaassociated protein 2 ; ia2 , insulinomaassociated protein 2 ; ica512 , islet cell autoantigen 512 ; pc1/3 , prohormone convertase 1/3 ; pc2 , prohormone convertase 2 ; ptprn , protein tyrosine phosphatase , receptor type , n ; ptprn2 , protein tyrosine phosphatase , receptor type , n polypeptide 2 ; sgiii , secretogranin iii ; snap25 , synaptosomalassociated protein 25 ; vamp2 , vesicleassociated membrane protein 2 .
the enhanced intracellular atp levels resulting from glucose metabolism inactivate atpsensitive k ( katp ) channels , causing membrane depolarization .
this triggers ca influx through voltagegated ca ( cav ) channels and subsequent soluble nethylmaleimidesensitive factor attachment protein receptor ( snare)mediated fusion of sgs .
depolarization also activates voltagegated k ( kv ) channels , which act to reestablish the resting membrane potential and thus suppress insulin secretion .
the inwardly rectifying k ( kir ) channel 6.2 and its associated sulphonylurea receptor ( sur)1 constitute the main katp channel responsible for glucoseinduced depolarization of cells . while kir6.2 associated with membrane rafts in cardiomyocytes , kir6.2 and sur1
however , kv2.1 and cav1.2 , and the snare proteins syntaxin 1a , synaptosomalassociated protein 25 ( snap25 ) and vesicleassociated membrane protein 2 ( vamp2 ) were all partially associated with membrane rafts ( figure  1b ) .
mcd , which caused their redistribution out of raft fractions , increased gsis , a result which capacitance measurements indicated was due , in part , to enhanced refilling of the readily releasable sg pool .
kv channel current amplitude was reduced in depleted cells , suggesting that membrane raft disruption promoted insulin secretion by hindering repolarization . in this context , it is noteworthy that snare proteins , in addition to their fundamental role in granule fusion , have also been reported to regulate katp , kv and cav channels .
thus , membrane rafts appear to contribute to the modulation of cell excitability , either by directly regulating channel activity or by spatially coordinating snarecoupled channel gating .
upregulated gsis and enhanced depolarization were also observed in mcdtreated ins1 cells , as was increased cortical factin remodeling .
these results were attributed to altered membrane phosphatidylinositol 4,5bisphosphate ( pip2 ) levels , as mcd increased pip2 hydrolysis whereas cholesterol loading appeared to enhance membrane pip2 levels and disrupt glucosedependent hydrolysis .
its accumulation impaired insulin secretion by reducing the sensitivity of katp channels to atp and stabilizing the cortical factin cytoskeleton .
this work suggests that the effects of cholesterol accumulation on gsis are coupled in part through pip2 .
possibly , as triton x100 insoluble raft fractions were reportedly enriched in pip2 . however , it is still unclear how this might influence glucosedependent pip2 hydrolysis or the regulation of katp channels comprised of kir6.2 and sur1 , both of which appear to be excluded from membrane rafts in cells .
in contrast to the observations that cholesterol depletion enhances gsis , further work by xia et  al . indicated that the inhibition of cholesterol biosynthesis in min6 insulinoma cells , which also disrupted the raft partitioning of cav1.2 , kv2.1 and snare proteins , impaired gsis . in this case , cav , kv and katp channel activity
similar results were reported in cells after cholesterol overloading , suggesting that these channels are sensitive to any deviations from optimal cholesterol levels , regardless of direction . as reducing kv and
katp channel activity promotes secretion , the component of impaired gsis resulting from altered channel function was attributed in these studies to the observed deficit in cav channel activity .
alterations of the membrane lipid environment also disrupted insulin secretion by dispersing ca influx independent of changes in channel activity or sg release competence .
this spatial uncoupling of ca influx and sgs has been proposed to hinder fusion pore formation , thus reducing the frequency of full fusion events and the efficiency of insulin release .
in addition , reduced sg exocytosis , detected in the absence of altered channel function , was a primary deficit in the cells of abca1islets .
the core snare fusion complex is comprised of the target ( t)snares syntaxin 1a , and snap25 on the plasma membrane and the vesicle ( v)snare vamp2 on vesicles . as previously described in neurons , cholesterolsensitive
syntaxin  1 and snap25 clusters define sites where sgs preferentially dock and fuse . in insulinoma cells ,
mcd dispersed syntaxin  1 and induced a redistribution of snap25 to the cytosol , both of which were accompanied by reductions in the number of docked sgs and gsis .
interestingly , diffusion of syntaxin  1a from granule docking sites and reduced gsis were also observed in ins1 cells cultured in high glucose , a condition that downregulated cholesterol biosynthesis and disrupted syntaxin  1acontaining membrane rafts .
however , studies demonstrating cholesteroldependent tsnare clustering did not always detect snare proteins in membrane raft fractions .
in addition , syntaxin  1a clustering was detected in proteoliposomes that did not support the formation of raftlike liquid ordered ( lo ) domains .
when cell snare proteins did partition with drms , a substantial pool was also detected in soluble fractions . in neuroendocrine cells ,
more recently , these proteins have been shown to form two conformationally distinct and spatially segregated tsnare dimer intermediates , only one of which supports fusion .
in addition , crosslinked complexes containing syntaxin  1a and the sec1/munc18like ( sm ) protein mammalian uncoordinated  18 ( munc18 ) , an accessory protein essential for secretion that participates in multiple stages of snare assembly and fusion , were restricted to nonraft fractions .
although these observations suggest that snare assembly and fusion might proceed in nonraft membrane regions , more work is required to understand the role of membrane rafts in the multiple , and often spatially and temporally distinct , stages of snare assembly and function .
proper sg biogenesis is an obvious prerequisite for secretion , and cholesterol , which is highly enriched in the granule membrane , is critical for their formation and integrity . in neuroendocrine cells ,
disruption of cholesterol biosynthesis with lovastatin , a 3hydroxy3methylglutarylcoenzyme  a ( hmgcoa ) reductase inhibitor , blocked the formation of granules from the transgolgi network ( tgn ) and caused swelling of the tgn cisterna . in min6 cells ,
lovastatin decreased sg numbers while increasing their average size , and this corresponded with reduced insulin content and impaired regulated secretory response .
disruption of either lanthosterol5desaturase ( sc5d ) or 7dehydrocholesterol reductase ( dhcr7 ) , the enzymes responsible for the last two steps in cholesterol synthesis , decreased pancreatic exocrine and endocrine sg numbers , and increased granule size .
studies in model membranes demonstrated that lanthosterol enhanced membrane flexibility while reducing intrinsic membrane curvature relative to cholesterol , suggesting that the altered sg biogenesis in sc5d mice resulted from the accumulation of lanthosterol in granule membranes .
cholesterol accumulation in abca1 islets was also suggested to disrupt sg biogenesis , a conclusion based on observations that cell golgi ultrastructure was altered and circulating proinsulin levels were enhanced .
however , although the insulin sgs of these mice were described to be  heterogeneous with respect to mean diameter compared with controls , no significant changes in sg numbers , size or distribution were observed .
enlarged sgs and impaired gsis downstream of depolarization were also observed in islets and insulinoma cells lacking the cholesterol transporter atpbinding cassette transporter  g1 ( abcg1 ) . in cells ,
abcg1 was reportedly expressed on sgs , and its loss led to reductions in sg cholesterol levels without altering total cellular or circulating cholesterol levels .
therefore , abcg1 has been proposed to maintain sg integrity by facilitating the retention of cholesterol within the granule inner membrane leaflet , thus counteracting the carriermediated diffusion of cholesterol from the sg outer membrane leaflet .
in addition to reducing sg membrane integrity , the dispersal of this pool could alter raftdependent processes elsewhere  a possibility supported by the observation that mcd extracted more cholesterol from cells lacking abcg1 .
this apparent redistribution of cholesterol to the plasma membrane might explain the latestage deficit in secretion ( i.e. downstream of ca influx ) detected in these islets , although it is tempting to speculate that a corresponding reduction of sg membrane cholesterol might have also perturbed raftassociated sg proteins , such as vamp2 .
whatever the case , this work suggests a unique mechanism of cell dysfunction that could be of particular interest given the observation that abcg1 expression is reduced in diabetic mice .
although the mechanisms governing this process are still debated , membrane rafts appear to play a role ( figure  1c ) .
prohormone convertase  1/3 ( pc1/3 ) and 2 ( pc2 ) , two cargo endoproteases responsible for the conversion of proinsulin to insulin during sg maturation , both partitioned with drms in neuroendocrine cells .
in addition , pc1/3 associated with the luminal leaflet of sgs in a cholesterolsensitive manner , and deletion of its membranebinding domain led to missorting of pc1/3 and its constitutive release . the membrane raft association of pc2 , in contrast , appeared to be sphingolipiddependent .
inhibition of sphingolipid synthesis with fumonisin blocked its targeting to sgs , as did truncation of its cterminal raftbinding domain .
the insulin processing enzyme carboxypeptidase  e ( cpe ) was also reported to associate with membrane rafts in sg membranes purified from pituitary .
cholesterol depletion dissociated cpe from sg membranes , and its sorting to sgs was disrupted in lovastatintreated att20 pituitary cells .
a similar missorting was observed in neuroendocrine cells when the cterminus of cpe , which mediated its partitioning with drms , was removed .
it should be noted , however , that cpe did not partition with raft fractions in min6 cells .
cholesterol appears essential for targeting the granin protein , secretogranin  iii ( sgiii ) , to immature granules , as it bound to sglike liposomes and isolated sg membranes in a cholesteroldependent manner , and deletion of its nterminal membranebinding domain caused its constitutive secretion .
in ins1 cells , sgiii binds cpe , and its interaction with chromogranin  a ( cga ) , another granin that promotes prohormone aggregation in the acidic luminal milieu of sgs , targeted cga to insulin sg membranes .
therefore , sgiii has been described as a multifunctional adaptor that facilitates the retention of prohormone aggregates in the cholesterolenriched membranes of nascent sgs , thus promoting their processing and maturation by raftassociated convertases .
this does not appear to be the case , as it was soluble in triton x100 and several other detergents typically used to designate raft association .
the transmembrane sg protein phogrin , additionally referred to as insulinomaassociated protein 2 ( ia2 ) or protein tyrosine phosphatase , receptor type , n polypeptide  2 ( ptprn2 ) , was also shown to interact with cpe , and this facilitated the targeting of both proteins to granules .
phogrin and its paralog , ica512 , like sgiii were also primarily soluble in triton x100 .
however , ica512 partitioned to a greater extent with drms after solubilization in lubrol ( dirkx r and solimena m , unpublished data , 2007 ) , a finding similar to that reported previously for vamp2 .
these results suggest that sg membranes , although highly enriched in cholesterol and sphingolipids , still appear to be organized in heterogeneous functional membrane raft domains with distinct protein profiles .
cholesterol is essential for proper cell function , and despite discrepancies regarding the effect of cholesterol depletion on some aspects of gsis , there now appears to be a clear consensus that accumulation of cellular cholesterol impairs insulin secretion ( figure  2 ) . in the end , however , the question remains  do membrane rafts regulate gsis ?
conceivably yes , as compelling evidence indicates that numerous proteins essential for granule biogenesis and insulin secretion associate with these domains in a cholesteroldepletion sensitive fashion .
however , at this time there is little evidence that physiological stimuli that trigger or augment insulin secretion ( e.g. glucose , glucagonlike peptide 1 etc . )
do so by altering the partitioning of these proteins in or out of membrane rafts .
in addition , although a recent study suggested that hyperglycemiainduced reductions in cholesterol might impair gsis in part by disrupting membrane rafts , it has not been clearly demonstrated that physiopathological conditions that induce cholesterol accumulation also alter membrane rafts .
for these reasons , it is difficult to assess at this time if membrane raft perturbation is a key factor contributing to cell dysfunction in type  2 diabetes .
thus , while it remains an appealing possibility , the hypothesis that impaired cellular cholesterol homeostasis disrupts insulin secretion by altering cell membrane rafts remains to be proven . 
a summary of the reported effects of cholesterol perturbation in pancreatic islets , cells or insulinoma cells .
arrows indicate increase (  ) or decrease (  ) in the described effect .
those effects associated with enhanced or reduced gsis are listed in green or red , respectively .
as noted below , exocytosis is listed only when observed directly ( i.e. changes in cadependent exocytotic response monitored by capacitance measurements taken independent of voltagegated ca [ cav ] channeldependent ca influx ) . * the deficit in regulated secretion associated with the sc5dmice
was reported from exocrine cells . * * although no net change in cholesterol was reported in abcg1 islets , secretory granule ( sg ) cholesterol levels were reduced , whereas plasma membrane levels appeared to be elevated . * * * although not measured directly , impaired exocytosis likely accounted in part for the secretory deficit in abcg1 islets , as glucoseinduced ca influx was unchanged compared with controls .
abca1 , atpbinding cassette transporter a1 ; abcg1 , adenosine triphosphatebinding cassette transporter g1 ; drm , detergent resistant membrane ; gck , glucokinase ; hmgcoa , 3hydroxy3methylglutarylcoenzyme a ; katp , adenosine triphosphatesensitive k channel ; kv , voltagegated k channel ; ldlr , low density lipoprotein receptor ; mcd , methylcyclodextrin ; pip2 , phosphatidylinositol 4,5bisphosphate ; sc5d , lanthosterol5desaturase ; snap25 , synaptosomalassociated protein 25 ; vamp2 , vesicleassociated membrane protein 2 .
 abstractthe failure of pancreatic cells to supply insulin in quantities sufficient to maintain euglycemia is a hallmark of type  2 diabetes . 
 perturbation of cell cholesterol homeostasis , culminating in elevated intracellular cholesterol levels , impairs insulin secretion and has therefore been proposed as a mechanism contributing to cell dysfunction . 
 the manner in which this occurs , however , is unclear . 
 cholesterol is an essential lipid , as well as a major component of membrane rafts , and numerous proteins critical for the regulation of insulin secretion have been reported to associate with these domains . 
 although this suggests that alterations in membrane rafts could partially account for the reduction in insulin secretion observed when cell cholesterol accumulates , this has not yet been demonstrated . in this review 
 , we provide a brief overview of recent work implicating membrane rafts in some of the basic molecular mechanisms of insulin secretion , and discuss the insight it provides into the cell dysfunction characteristic of type  2 diabetes . 
 ( j diabetes invest , doi : 10.1111/j.20401124.2012.00200.x , 2012 ) 
Introduction
Cholesterol Homeostasis and Cell Dysfunction
Glucose Uptake and Metabolism
Cell Channel Function and Secretory Granule Exocytosis
Granule Biogenesis: Cholesterol and Secretory Granule Membrane Integrity
Granule Biogenesis: Protein Sorting to Secretory Granules
Conclusions