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doi:10.1152/ajpendo.90538.2008 296:1-10, 2009. First published Sep 23, 2008;Am J Physiol Endocrinol Metab

Taila Hartley, John Brumell and Allen Volchuk

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Review

Emerging roles for the ubiquitin-proteasome system and autophagyin pancreatic �-cells

Taila Hartley,1,2 John Brumell,3,4,6 and Allen Volchuk1,2,5

1Division of Cell and Molecular Biology, Toronto General Research Institute, University Health Network, Toronto;Departments of 2Biochemistry, 3Molecular Genetics, 4Institute of Medical Science, and 5Physiology, University of Toronto;and 6Cell Biology Program, The Hospital for Sick Children, Toronto, Ontario, Canada

Submitted 24 June 2008; accepted in final form 12 September 2008

Hartley T, Brumell J, Volchuk A. Emerging roles for the ubiquitin-proteasome system and autophagy in pancreatic �-cells. Am J Physiol Endo-crinol Metab 296: E1–E10, 2009. First published September 23, 2008;doi:10.1152/ajpendo.90538.2008.—Protein degradation in eukaryotic cells is me-diated primarily by the ubiquitin-proteasome system and autophagy. Turnover ofprotein aggregates and other cytoplasmic components, including organelles, isanother function attributed to autophagy. The ubiquitin-proteasome system andautophagy are essential for normal cell function but under certain pathologicalconditions can be overwhelmed, which can lead to adverse effects in cells. In thisreview we will focus primarily on the insulin-producing pancreatic �-cell. Pancre-atic �-cells respond to glucose levels by both producing and secreting insulin. Theinability of �-cells to secrete sufficient insulin is a major contributory factor in thedevelopment of type 2 diabetes. The aim of this review is to examine some ofthe crucial roles of the ubiquitin-proteasome system and autophagy in normalpancreatic �-cell function and how these pathways may become dysfunctionalunder pathological conditions associated with metabolic syndromes.

autophagy; insulin secretion; ubiquitin proteasome; endoplasmic reticulum stress

THE REGULATION OF BLOOD GLUCOSE is accomplished by severalorgans that maintain appropriate levels during fasting as wellas in postprandial states. In the latter case, the pancreas,specifically the pancreatic �-cells found in the islets of Lang-erhans, is essential for blood glucose disposal. The �-cell is theonly source of circulating insulin, which is essential for bothstimulating peripheral tissue glucose uptake and inhibitingliver glucose production, among other metabolic effects (112).Glucose is the main metabolite responsible for regulatinginsulin secretion from the �-cell (3), although other metabo-lites such as free fatty acids can also stimulate secretion and theprocess can be modulated by incretin hormones such as glu-cagon-like peptide 1 (24, 88). In addition to stimulating insulinsecretion, glucose also stimulates insulin production, both byincreasing insulin gene transcription and by increasing insulintranslation (Fig. 1) (79, 116, 121, 122). Pancreatic �-cells arecentral to blood glucose regulation, and if they are destroyedby the immune system or are either dysfunctional or signifi-cantly depleted then type 1 or type 2 diabetes, respectively, canresult. As a secretory cell that must secrete large amounts ofinsulin this particular cell type is particularly susceptible tocertain stresses.

Even under fasting glucose levels an estimated 40–50% oftotal cellular protein produced by purified rat �-cells is insulinand stimulatory glucose levels acutely increase insulin biosyn-thesis (107). To accommodate this large demand the �-cell has

evolved an extensive endoplasmic reticulum (ER) (25). It istherefore not unexpected that the �-cell is very sensitive toconditions in which the ER protein folding capacity is notsufficient to meet protein folding demands. Such a state isreferred to as ER stress and the cellular response to suchconditions is referred to as the unfolded protein response(UPR). The UPR is involved in sensing the protein foldingcapacity of the ER and eliciting cellular effects that reduce ERstress by transiently attenuating translation, increasing chaper-one expression, and increasing degradation of misfolded pro-teins (101). In mammalian cells this is mediated primarily bythree ER stress sensors (PERK, IRE-1, and ATF6) that sensethe level of misfolded protein in the ER and transmit down-stream signals. The PERK pathway via eIF2-� phosphoryla-tion transiently attenuates translation and induces gene expres-sion changes via upregulation of the transcription factor ATF4.IRE-1 signals by splicing the XBP-1 mRNA that results inenhanced XBP-1 translation, whereas ATF6 is activated byregulated proteolysis once it is trafficked to the Golgi. XBP-1and ATF6 regulate the induction of the majority of UPRresponse genes. The intricacies of ER stress sensing and thecomplement of responses mediated by these pathways is re-viewed in Ref. 101. It has recently been appreciated that as aconsequence of its high secretory protein production the pan-creatic �-cell must rely on an efficient UPR system to maintainER homeostasis (25, 29). Chronic or persistent ER stress canresult in the induction of apoptosis (92). Thus the �-cell maybe particularly susceptible to programmed cell death in condi-tions such as obesity and diabetes, where insulin demands arehigh.

Address for reprint requests and other correspondence: A. Volchuk, TorontoGeneral Research Institute, 101 College St., TMDT 10-707, Toronto, ON,M5G 1L7 Canada (e-mail: [email protected]).

Am J Physiol Endocrinol Metab 296: E1–E10, 2009.First published September 23, 2008; doi:10.1152/ajpendo.90538.2008.

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In addition to susceptibility to ER stress, the pancreatic�-cell is also known to be sensitive to oxidative stress. This islikely due to the fact that the �-cell expresses relatively lowlevels of major antioxidant enzymes including superoxidedismutase, catalase, and glutathione peroxidase that function toremove reactive oxygen species (34, 68, 119). Pancreatic�-cells are sensitive to conditions such as chronically elevatedglucose and free fatty acid levels that increase the amount ofreactive oxygen species within cells (99, 100). Such chronicconditions are known to cause adverse effects such as reducedinsulin expression and increased cellular damage and celldeath.

Both ER stress and increased oxidative stress can also leadto the accumulation of misfolded and/or aggregated proteins in

the cell (101, 108). Such material needs to be removed toprevent protein aggregates from accumulating to toxic levels.Two principal systems, the ubiquitin-proteasome system andautophagy, function to maintain cellular homeostasis by recy-cling macromolecules and in the latter case entire organelles byintracellular degradation. This review will examine the role ofboth systems in pancreatic �-cell physiology and the potentialconsequences that may result when they are overwhelmed ordo not function efficiently.

The Ubiquitin-Proteasome System in Pancreatic �-Cells

One of the main mechanisms for the degradation of cellularproteins is the ubiquitin-proteasome system. Proteins targeted

Fig. 1. Glucose stimulated insulin biosynthesis and secretion in pancreatic �-cells. Following preproinsulin mRNA transcription and processing, preproinsulinis synthesized in the rough endoplasmic reticulum (rER) and is converted to proinsulin in the lumen of the endoplasmic reticulum (ER). Proinsulin is folded andtransported to the Golgi apparatus. At the trans-Golgi network (TGN) proinsulin is sorted into secretory granules and is processed to mature insulin by the actionof prohormone-converting enzymes. A more detailed discussion can be found in Ref. 23. Glucose is transported across the plasma membrane through specificglucose transporters, primarily GLUT2 in rodent �-cells. Once in the cell glucose is phosphorylated and metabolized. In the �-cell glucose metabolism influencesboth acute insulin secretion (A) and insulin transcription and translation (B). Glucose-stimulated insulin secretion results from the increased ATP/ADP ratio uponoxidation of glucose by the mitochondria. The increased ATP/ADP ratio results in closure of the ATP-sensitive KATP channels. This in turn results indepolarization of the plasma membrane and opening of the L-type voltage-dependent Ca2� channel. Influx of extracellular Ca2� stimulates the fusion of insulingranules with the cell surface. Glucose induces insulin translation largely by promoting its initiation. Glucose promotes the recruitment of 40S ribosomes tomRNA through its effects on eIF2 and eIF4F (reviewed in Ref. 97). Glucose control of insulin transcription involves the generation of poorly characterized signalsfollowing glucose metabolism that activate downstream kinases that modulate various transcription factors involved in insulin gene transcription. Phosphati-dylinositol 3-kinase and stress-activated protein kinase 2 (SAPK2/P38) can induce a shift of the PDX-1 transcription factor from the cytoplasm to the nucleus,where it binds and activates the insulin gene promoter (97). Stimulatory glucose concentrations also activate other transcription factors such as E47, Beta2, andMafA, which also act by binding to insulin gene promoter regions. VDC channel, voltage-dependent calcium channel.

Review

E2 UBIQUITIN-PROTEASOME SYSTEM AND AUTOPHAGY IN �-CELLS

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for degradation are marked by ubiquitination and are degradedby the 26S proteasome. Three distinct enzymes are required tolink ubiquitin onto proteins: E1 (ubiquitin-activating enzyme), E2(ubiquitin-carrier or conjugating proteins), and E3 (ubiquitin-protein ligase), which recognizes a specific protein substrateand catalyzes the transfer of activated ubiquitin to the clientprotein. Because the ubiquitin-proteasome system can controlprotein abundance, the cellular functions are varied and diverseand include general protein degradation, regulation of genetranscription, maintenance of protein quality control, immunesystem regulation, and regulation of amino acid levels, amongmany other roles (19, 41, 83, 118). The mechanistic details ofubiquitin-proteasome-mediated protein degradation are de-scribed in the above reviews and will not be discussed furtherhere. The specificity for protein targeting for ubiquitin-protea-some degradation is mediated by the E3 enzymes, whichcomprise a large family of proteins (28, 39, 85, 105). Althoughthe protein targets of the E3 enzymes for the most part stillneed to be identified, several of the E3s have been implicatedin human diseases such as cancer, cardiovascular, autoimmu-nity, and other conditions (28, 33, 85, 105). Some E3 enzymesappear to be expressed at relatively high levels in islets and

�-cells on the basis of analysis of microarray expression dataavailable on internet databases (see Supplemental Table S1).However, biochemical analysis of the function and targets ofthe various E3 enzymes expressed in �-cells is lacking.

The role of the ubiquitin-proteasome system in pancreatic�-cells is turning out to be highly complex. In this section wewill discuss some recent studies examining the function of theubiquitin-proteasome system in maintaining insulin biosynthe-sis and secretion, in ER-associated degradation (ERAD), and inthe degradation of cytosolic protein aggregates and how pro-teasome dysfunction may contribute to pathogenesis in pan-creatic �-cells.

Ubiquitin-proteasome system and insulin biosynthesis, se-cretion, and signaling. Several recent studies have demon-strated an important role for the ubiquitin-proteasome systemin �-cell physiology including maintaining proper insulin lev-els and facilitating normal secretion (summarized in Fig. 2).Although it is clear that the ubiquitin-proteasome system isessential for maintaining insulin production and secretion,exactly how it is involved has yet to be worked out. Most of thestudies have used proteasome inhibitors and have monitoredinsulin biosynthesis and secretion. Proteasome inhibition with

Fig. 2. Summary of some of the roles of ubiquitin-proteasome system and autophagy in pancreatic �-cells. In addition to the general function of autophagyobserved in other tissues described in the text, autophagy in �-cells serves to dispose of aggregated proteins that accumulate in aggresome-like structures (ALIS)that result from chronic conditions such as elevated hyperglycemia, free fatty acids (FFA) and reactive oxygen species (ROS) (A), retire old insulin granules (B),and potentially relieve ER stress that can result from the accumulation of misfolded proteins (C). The ubiquitin-proteasome system has been shown to regulatethe activation of membrane channels such as the VDC channel and the KATP channel (I), as well as play a role in the regulation of proinsulin transcription (II),but how this occurs or what role this plays remains unclear. In addition, the ubiquitin-proteasome system is also the degradative machinery of the ER-associateddegradation system (ERAD) (III), which functions to eliminate misfolded proteins retrotranslocated from the ER lumen. The ubiquitin-proteasome system mayalso serve to degrade misfolded cytosolic and ER proteins that accumulate in pancreatic �-cells as a result of islet amyloid polypeptide (IAPP) deposition (IV)that is commonly found in islets of type 2 diabetic individuals. See text for details.

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lactacystin has been reported to reduce proinsulin synthesis inmouse islets, which seems counterintuitive given that protea-some inhibition might be expected to result in increased insulinlevels (58). This study, however, also reported that proteasomeinhibition induces ER stress, which may attenuate translationand indirectly reduce insulin biosynthesis.

Proteasome inhibition has also been shown to affect glucose-stimulated insulin secretion in �-cells, although different re-sults have been reported. Proteasome inhibition in mouse islets(58) and MIN6 cells (55) inhibits acute glucose-stimulatedinsulin secretion, whereas lactacystin enhanced acute glucose-stimulated insulin secretion in rat islets (71). The role of theubiquitin-proteasome system in the insulin secretion pathwayis likely to be complex since recent studies have shown that thesurface expression of molecules involved in regulated insulinsecretion such as the ATP-sensitive potassium (KATP) channel(124) and the activity of the voltage-dependent Ca2� channel(55) are dependent on the ubiquitin-proteasome system. Thelatter study has shown that the �-subunit of the voltage-dependent Ca2� channel undergoes ubiquitination and thatproteasome inhibition impairs channel activity. Thus the pro-teasome may control the abundance or activity of multipleproteins that regulate the complex process of glucose-stimu-lated insulin secretion (Fig. 1).

In addition, the ubiquitin-proteasome system may regulatethe function of other important pathways in the �-cell. Forinstance, insulin has been shown to promote ubiquitin-protea-some system mediated degradation of insulin receptor substrate2 (IRS2) in 3T3-L1 fibroblast cells, which can be blocked bythe 26S proteasome inhibitors MG132 or lactacystin (103). Asa result, this attenuates insulin signaling via IRS2. IRS2 iscritical for pancreatic �-cell function and cell survival (8). Itremains to be determined whether the levels of IRS2 arecontrolled by the ubiquitin-proteasome system in �-cells, butthe possibility exists that �-cell survival is dependent on theproteasomes’ ability to correctly regulate IRS2 levels undervarious external conditions.

Chronic cytokine exposure associated with chronic inflam-mation in obesity and diabetes has been implicated in causingdysfunction in several tissues including �-cells (26, 43). Somestudies have identified that cytokine signaling in pancreatic�-cells involves proteasomal degradation of key signalingcomponents. One study has shown that cytokines can increasecytosolic calcium levels, which leads to ubiquitination anddegradation of islet-brain 1/JNK interacting protein 1 (1).IB1/JIP1 normally prevents JNK activation, a signaling path-way implicated in induction of apoptosis in response to cyto-kines in �-cells (16). The ubiquitin-proteasome system is alsoimplicated in cytokine-induced activation of the NF-�B tran-scription factor, which also plays a role in the induction ofapoptosis. Cytokine-induced degradation of I�B� and activa-tion of NF-�B in islets and pancreatic �-cell lines can beinhibited by proteasome inhibitors (64). Although inhibitingthe proteasome itself is unlikely to be a useful therapeuticstrategy for reducing the cytotoxic effects of cytokines, target-ing the E3 ligases specifically responsible for ubiquitination ofIB1/JIP1 or I�B� may be.

The ubiquitin-proteasome also plays a role in cell prolifer-ation. The Skp2 ubiquitin ligase was recently shown to controlcellular p27 levels in �-cells, an important molecule involvedin �-cell proliferation (128). The identification of ubiquitin-

proteasome targets in pancreatic �-cells is only just beginningand it is likely that the activity or abundance of many keysignaling proteins will be shown to be intimately dependent onthis system.

Several genes involved in the ubiquitin-proteasome systemare downregulated in a mouse pancreatic �-cell line withimpaired insulin secretion, suggesting that defects in this sys-tem may lead to impaired secretion (81). Clearly more work isrequired to delineate the role of the ubiquitin-proteasomesystem in maintaining normal insulin biosynthesis and secre-tion in pancreatic �-cells as well as in regulating cellularsignaling pathways in this cell type.

Ubiquitin-proteasome system and ER stress. In addition tocontrolling the abundance of cytosolic proteins the ubiquitin-proteasome system is also required for degrading terminallymisfolded ER proteins via the ERAD system. As mentionedabove, owing to the requirement of �-cells to maintain insulinproduction this cell type is particularly sensitive to ER stress.In addition, conditions associated with obesity and the devel-opment of diabetes, such as chronically elevated cytokines,free fatty acids, and glucose, have been shown to cause ERstress in both in vivo and in vitro models (11, 54, 56, 66, 117).Induction of the UPR during times of ER stress results in atransient decrease in the overall amount of translation withinthe �-cell and an increase in the levels of ER chaperones (15,127). Interestingly, although ER stress leads to PERK-medi-ated translational inhibition, recovery from translational re-pression seems to be critical for �-cell function and survival(15, 127). Thus the �-cell must also likely upregulate ERADcomponents during ER stress. The ERAD system degradesterminally misfolded proteins by retrotranslocating them out ofthe ER into the cytosol where they are degraded by theubiquitin-proteasome system (57, 80). This may be importantfor maintaining normal insulin biosynthesis since proteasomeinhibition reduces proinsulin biosynthesis (58). Whether mis-folded insulin in normal �-cells or in obese or diabetic condi-tions is actually retrotranslocated to the cytosol for ubiquitin-dependent proteasome degradation requires experimental ver-ification. However, this appears likely since misfolded insulinin the Akita diabetic mouse induces expression of ERADcomponents, which are required for intracellular degradation ofthe misfolded mutant insulin (2).

Ubiquitin-proteasome system and islet amyloid. Anothersituation in which the ubiquitin-proteasome system may beimportant to �-cell dysfunction in diabetes involves islet amy-loid deposition. A pathophysiological feature of type 2 diabetesis islet amyloidosis in the pancreatic islet (46). The maincomponent of islet amyloid is islet amyloid polypeptide(IAPP), also known as amylin (14, 51, 73, 120). IAPP is anormal product of the pancreatic islet �-cell and is stored alongwith insulin in secretory granules. Islet amyloid localizes toareas of cell degeneration and the process of amyloidosis hasbeen associated with progressive loss of pancreatic �-cell massby apoptosis (9, 46, 50, 72). The reason why islet amyloiddeposits form is not completely understood, although it mayinvolve impaired processing of the IAPP precursor moleculethat triggers the process. Pancreatic �-cell toxicity is likely dueto oligomerizing propensity of IAPP intermediates, rather thanthe mature fibrils (20, 49, 77).

The mechanism by which amyloid deposits cause cell deathis not entirely understood, although some recent work has been

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suggestive of several potential effects. A property of IAPPoligomers closely associated with cytotoxicity is the formationof nonselective membrane channels (82). In addition, bothextracellular and intracellular amyloidosis have been shown tocause ER stress and an associated increase in ubiquitinatedproteins (12, 45). Overexpression of human IAPP (hIAPP) thathas a propensity to aggregate in mice caused ER stress activa-tion including increased expression of spliced Xbp-1 (45).Human islets treated with hIAPP showed increased ERstress and decreased proteasome activity, resulting in theaccumulation of ubiquitinated proteins (12). These effectsmay contribute to apoptotic cell death if as a result of ERstress the UPR is unable to restore ER function and theaccumulation of unfolded proteins overwhelms the protea-some, leading to decreased proteasome activity (4, 76). Thuschronic ER stress combined with insufficient degradation bythe ubiquitin-proteasome may trigger apoptotic pathwaysleading to cell death (92).

Ubiquitin-proteasome system and cellular protein aggre-gates. Several cellular stresses including heat shock and agentsthat increase mitochondrial and oxidative stress can causeprotein misfolding and the accumulation of cytosolic proteinaggregates (62, 108). If the accumulation of cytosolic proteinaggregates exceeds disposal the aggregates can be stored incytosolic structures called aggresomes (62). Aggresomes formfrom small protein aggregates that are targeted along microtu-bules to the microtubule organizing center, where they coa-lesce to form a larger structure. Material targeted to aggre-somes is degraded by the ubiquitin-proteasome system, butalso by autophagy (discussed below). In addition to aggre-somes, other structures referred to as aggresome-like structures(ALIS) have been discovered that result from accumulation ofmisfolded proteins or aborted translation products (67, 110).The clearance of aggregated material is dependent on both theubiquitin-proteasome system and autophagy (18, 30, 110).Such structures have recently been observed to accumulate inchronic conditions associated with diabetes in pancreatic�-cells, but they are likely degraded mainly via the autophagysystem (discussed below) (53).

Autophagy in Pancreatic �-Cells

In addition to the ubiquitin-proteasome system, cells alsorely on autophagy for the degradation of cellular components.Autophagy is a degradative system that is executed by twoubiquitin-like protein conjugation reactions: Atg5 is conju-gated to Atg12, which then forms a complex with Atg16L1.This complex acts as an E3 ligase to promote the conjugationof LC3 (a homologue of yeast Atg8) to the phospholipidphosphatidylethanolamine (36, 47, 87). Lipid-conjugated LC3is thought to promote membrane interactions (tethering andhemifusion), promoting the completion of autophagosome for-mation and/or their fusion with lysosomes (84, 109). TheAtg5-12-16L1 complex is thought to localize LC3 conjugationto sites where autophagy occurs (32), and this targeting may beregulated by members of the Rab GTPase family (48). BecauseLC3 is covalently attached to membranes involved in autoph-agy, it serves as a powerful marker to visualize the autophagyprocess (52). Upstream regulators of autophagy in mammaliancells include class III PI3-kinase VPS34, which is part of acomplex with the tumor suppressor Beclin-1 (10), and the

kinases ULK-1/2 (38). The mechanism of autophagosomeformation and the source of membrane required for autophagyare not clear, possibly complicated by the fact that autophagyis a heterogeneous process (96). Indeed, there are differentvariations of autophagy depending on the organelle or sub-strates being degraded. In all cases autophagy terminates withthe delivery of cytoplasmic cargo into the lysosome for deg-radation and recycling of macromolecular components. Severalrecent reviews deal with the molecular details of these complexpathways (59, 69).

It should also be mentioned that in addition to degradation oflong-lived proteins and elimination of cellular organelles, au-tophagy is used to produce amino acids under nutrient-depletedconditions. Insulin signaling, which enhances protein synthe-sis, also inhibits autophagy in insulin target tissues (17, 78).Thus there is the potential for alterations in this control ininsulin-resistant states such as diabetes (78). In this review wewill focus on the current understanding of what is known aboutthe role of autophagy in pancreatic �-cells. We will highlightrecent findings on the function of autophagy in the ER stressresponse, in potential degradation of aggregated proteins, andin the degradation of insulin granules.

Autophagy and the degradation of cytosolic proteins. Al-though the ubiquitin-proteasome system is involved in thedegradation of numerous cytosolic proteins and those retro-translocated from the ER, some studies have shown that certainproteins are not degraded by this system (37, 60, 94, 95).Inhibiting the proteasome has been shown to activate autoph-agy, suggesting that the two are functionally coupled and canact in a compensatory fashion to one another or autophagy mayserve as a backup system for proteasomal degradation (21). Inaddition, autophagy does not seem to be limited to bulkdegradation. Increasing evidence supports the idea that auto-phagy can act in a selective manner. Indeed, autophagy can bethe preferred mechanism for the degradation of specific pro-teins (90). Ding and Yin (22) have recently reviewed thecurrent understanding on how misfolded protein degradation isdivided between autophagy and the ubiquitin-proteasomesystem.

As mentioned previously, several cellular stresses can causethe accumulation of cytosolic protein aggregates. The clear-ance of some of this aggregated material has been shown to bedependent, at least in part, on the autophagy pathway (30). Indiabetes chronic hyperglycemia induces elevated reactive ox-ygen species production (99, 100). Recently, we have discov-ered that chronic hyperglycemia induces the appearance ofubiquitinated-protein aggregates in a pancreatic �-cell line(INS-1 832/13) (53). Such ubiquitinated protein aggregates arealso present in �-cells (and other cell types) in the Zuckerdiabetic fatty rat during the development of diabetes (53).Treatment of INS1 832/13 pancreatic �-cells with 3-MA (aninhibitor of autophagy), but not epoxomicin (an inhibitor of theproteasome) induced the appearance of ubiquitinated aggre-gates. Thus, at least in this context, the disposal of misfolded oraggregated protein appears to be mediated by autophagy. Whysuch ubiquitinated protein aggregates accumulate during hy-perglycemia is not known, although it is possible that thesestructures are storage sites for nonfunctional proteins duringtimes of stress. These are then removed via the autophagypathway once cellular conditions improve. In the �-cell theproteins targeted to the ubiquitinated protein aggregates still

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need to be identified. It is possible that insulin is retrotranslo-cated to the cytosol or that autophagy is responsible fordegrading areas of the ER that have accumulated and seques-tered such aggregates.

How ubiquitinated protein aggregates are targeted for auto-phagy-mediated degradation has yet to be established. In thiscontext it would be interesting to determine whether p62, whichis targeted to cytoplasmic ubiquitin-containing protein aggregatesin some cells (7), is present in these structures in �-cells. p62interacts with the autophagosome marker protein LC3 and withproteins containing K63-linked polyubiquitin and may facili-tate autophagy-mediated clearance of cellular inclusions (61,111). p62 has recently been shown to be expressed in �-cells(65), but its intracellular distribution and function in the �-cellwas not examined.

In addition to chronic hyperglycemia, diabetes associatedwith obesity is characterized by elevated circulating free fattyacids. Free fatty acids are known to induce apoptosis inpancreatic �-cells in vitro (16). In one study with isolatedhuman islets, chronic exposure to high levels of free fatty acidsshowed clear examples of autophagy in electron microscopicimages (74). Whether or not the upregulated autophagy iscontributing to promoting survival in this case or is itself acausative factor in promoting apoptosis remains to bedetermined.

Autophagy, ER stress, and degradation of ER components.Misfolded protein accumulation in the ER has traditionallybeen thought to be removed primarily by the ERAD systemthat is induced by the UPR (101). Recently, however, theimportance of autophagy in the ER stress response has beenestablished (6, 44). In yeast the process may involve a novelform of autophagy specifically induced by ER stress thatresults in the formation of autophagosomes that include ERmembranes (5, 6). Interestingly, these structures appear toselectively sequester portions of the ER but do not seem to beinvolved in degrading the sequestered material. It will beinteresting to determine whether similar structures occur inmammalian cells and whether they differ from more traditionalautophagosomes involved in degrading macromolecules.

There are several examples of mutant versions of membraneor secretory proteins that aggregate in the ER and appear torequire the autophagy system for degradation. Expression of amutant version of a protein called dysferin present in musclecells induced autophagy, which was required to degrade dys-ferin aggregates (31). This shows that in some cases aggre-gated proteins in the ER can be degraded by autophagy, whichmay act in conjunction with the ERAD system. Anotherpathological example where autophagy may play a prominentrole is familial neurohypophyseal diabetes insipidus (13). In acell culture model expression of a mutant version of thesecretory protein vasopressin causes an accumulation of theprotein in the ER and induces markers of autophagy (13).Inhibition of autophagy results in increased cell death, suggest-ing that autophagy activation acts as a prosurvival mechanism.Another example is �1-antitrypsin deficiency, which resultsfrom a mutant form of this protein that tends to aggregate in theER of liver cells, preventing its normal secretion (93). A seriesof studies have determined that autophagy is a critical systemthat is used for the degradation and removal of mutant �1-antitrypsin (93). In all of these cases it is aggregated proteinthat appears to require autophagy for degradation, whereas

soluble misfolded protein is likely disposed of by the ERADpathway.

Although not all forms of aggregated protein accumulationthat cause ER stress necessarily activate the UPR (42), there isa link between the UPR signaling pathways and autophagyinduction. In mammalian cells some studies have shown thatinduction of autophagy requires the JNK kinase (70, 126). JNKcan be activated by the IRE-1 pathway of the UPR (115).Recent studies have shown that IRE-1 is required for autoph-agy induction in ER-stressed mammalian cells (21, 89). Theaccumulation of LC3-positive vesicles can be triggered by theER stress-inducing compounds tunicamycin or thapsigargin,which is dependent on IRE1 but not PERK or ATF6 (89). Inaddition, studies in yeast have shown that the IRE1 pathwaycan regulate autophagy induction in that cell (125). However,the precise involvement of the UPR pathways in autophagyinduction remains to be sorted out as some recent studies haveconcluded that the PERK-eIf2� pathway also has an importantrole in inducing autophagy (31, 63). ER stress has also beenshown to lead to the release of calcium from the ER into thecytosol, and calcium can in turn activate various kinases andproteases potentially involved in autophagy (44).

eIF2� has also been implicated as an autophagy mediator inseveral other models unrelated to ER stress per se (104), but tothe integrated stress response (101). Viral infection, amino acidstarvation, and heme depletion are all thought to induce auto-phagy through other kinases that phosphorylate eIF2� such asprotein kinase R, general control nonderepressible-2 (GCN2),and heme-regulated inhibitor (HRI).

The role of autophagy in the ER stress response in pancreatic�-cells has not yet been reported. Although acute ER stressresponse may not require autophagy, it is possible that duringtimes of chronic ER stress activated by chronic conditions(elevated glucose, cytokines, and free fatty acids), the autoph-agy system may be required to deal with an overwhelmingaccumulation of misfolded or aggregated proteins.

Autophagy and the degradation of insulin granules. Inresponse to elevated glucose insulin is secreted by the exocy-tosis of granules at the cell surface and enhanced insulintranslation is mainly responsible for replenishing the lost in-sulin (114). Interestingly, insulin synthesis is maintained evenunder nonstimulatory glucose concentrations, indicating thatthe cell has a natural bias toward continual replenishment ofinsulin stores (3, 98, 107). However, �-cells contain a rela-tively constant number of secretory granules whose half-life is�3–5 days (35, 102). Thus insulin granules must undergodegradation to maintain granule numbers relatively constant.Early studies determined that granule degradation in �-cellsoccurs by a process called crinophagy, the fusion of insulingranules with lysosomes (35, 91, 106). More recent studieshave suggested that autophagy is also responsible for thedegradation of insulin granules (114). Studies in Rab3A-defi-cient mice, which have defective insulin secretion, have shedsome insight into the role of autophagy in maintaining granulenumber (75). Despite the secretion defect, insulin biosynthesisremains normal in these animals and hence a net overproduc-tion occurs. However, total insulin content remains relativelynormal in Rab3A(�/�) mouse islets owing to a markedupregulation in autophagic degradation of insulin granules.This suggests that autophagy can play a protective role in�-cells during signaling disconnection between insulin synthe-

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sis and secretion (75). Interestingly, these authors also foundthat there was a marked decrease in the expression of thelysosomal protein LAMP-2 in the islets of these mice.LAMP-2, which is a known to play a significant role inautophagy (27, 86), may be a potential regulator of granuledegradation in �-cells.

The role of autophagy in normal �-cell physiology and theregulation of this process to maintain �-cell granule numberrequires further investigation. Similarly, it is possible thatdefects in the regulation of autophagy occur in diabetes. Thismay contribute to imbalanced insulin content and as a resultreduced secretion capacity. In addition, a recent study hasshown that autophagy is important in the degradation of mito-chondria in �-cells and this process may be dysfunctional indiabetes (113).

Conclusion and Future Directions

Although both the ubiquitin-proteasome system and autoph-agy have already been shown to mediate important functions inthe pancreatic �-cell, the repertoire of cellular pathways thesesystems control is only beginning to be uncovered. These areessential systems for controlling protein and organelle abun-dance and are likely to be central in modulating many aspectsof �-cell function. Future studies using �-cell-specific knock-out models of important genes involved in the ubiquitin-proteasome system and autophagy pathways should providemore detail into the major cell pathways (signaling, secretion,cell apoptosis, etc.) that these pathways control and the mech-anisms involved.

In addition the study of these systems in the context ofdiabetes is also just beginning. There are likely to be defects inthe ubiquitin-proteasome system and/or autophagy in diabetesthat may adversely affect �-cell function. Such defects maylead to the inappropriate accumulation of dysfunctional mito-chondria, insulin granules, or activation of apoptotic pathways.Furthermore, it is becoming apparent that cellular stress ofvarious forms can induce dysfunctional protein folding, whichcan cause the accumulation of misfolded and aggregated pro-teins in cells (40, 123). The inability of the ubiquitin-protea-some system and autophagy pathways to clear protein aggre-gates may be intimately related to cell death in diseases such asdiabetes. The future study of these systems in the context ofdiabetes should uncover the defects that arise and potentialmethods to improve the function of these systems to potentiallycounteract the detrimental effects of diabetes on pancreatic�-cell function.

Note Added in Proof

While this manuscript was in press, two studies appearedshowing an essential role of autophagy in maintaining pancre-atic �-cell mass and function and protection against diabetes inmouse models (Jung HS, Chung KW, Won Kim J, Kim J,Komatsu M, Tanaka K, Nguyen YH, Kang TM, Yoon KH,Kim JW, Jeong YT, Han MS, Lee MK, Kim KW, Shin J, LeeMs. Cell Metab 8: 318–324, 2008; Ebato C, Uchida T, Ara-kawa M, Komatsu M, Ueno T, Komiya K, Azuma K, Hirose T,Tanaka K, Kominami E, Kawamori R, Fujitani Y, Watada H.Cell Metab 8: 325–332, 2008; and Meijer AJ, Codogno P. CellMetab 8: 275–276, 2008).

ACKNOWLEDGMENTS

We thank Dr. Michael Wheeler and Dr. Hongfang Lu for comments on themanuscript.

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