Proinsulin misfolding is an early event in the progression to type 2 diabetes
The progression of β-cell dysfunction with endoplasmic reticulum ( ER ) stress in prediabetes , to ß-cell failure with full-blown type two diabetes ( T2D ) , has long been the subject of intensive investigation ( Fonseca et al . , 2011; Rabhi et al . , 2014 ) . What is observed is that a chronic increase in insulin secretory demand over time leads to depletion of ß-cell secretory granule reserves despite a dramatic compensatory increase in secretory pathway activity ( Alarcon et al . , 2016; Rustenbeck , 2002 ) . By electron microscopy , pancreatic sections of human subjects with T2D show a marked increase of ER volume density in β-cells with an induction of ER stress markers triggered by high glucose exposure ( Marchetti et al . , 2007 ) . Similar indicators of ER stress have been established in the β-cells of animal models ( Yang et al . , 2016 ) , such as leptin receptor-deficient diabetic mice [known as LepRdb/db ( Diani et al . , 1984; Laybutt et al . , 2007; Like and Chick , 1970 ) that develop insulin resistance progressing to T2D , which is linked to overeating . Hypersynthesis of proinsulin ( Arunagiri et al . , 2018; Back et al . , 2009 ) is a condition proposed to increase proinsulin misfolding ( Liu et al . , 2005; Scheuner et al . , 2005 ) — which can promote ER stress with abnormal ß-cell ER expansion — whereas suppression of proinsulin protein synthesis actually alleviates ß-cell ER stress ( Szabat et al . , 2016 ) . Insulin-deficiency caused directly by proinsulin misfolding has been proved unequivocally in an autosomal-dominant form of diabetes known as Mutant INS-gene-induced Diabetes of Youth ( MIDY ) ( Liu et al . , 2010b ) , which occurs in patients bearing one mutant and one wild-type INS allele ( Liu et al . , 2015; Støy et al . , 2010 ) . The disease in humans is pathogenetically identical to that seen in the mutant Akita diabetic mouse ( Izumi et al . , 2003 ) or Munich MIDY Pig ( Blutke et al . , 2017 ) – which are animals expressing one mutant INS allele encoding proinsulin-C ( A7 ) Y that is quantitatively misfolded due to an inability to form the Cys ( B7 ) -Cys ( A7 ) disulfide bond . Ordinarily the expression of only one WT INS allele would be sufficient to avoid diabetes , but Akita mice develop diabetes despite expressing three alleles encoding WT proinsulin in addition to the one encoding mutant proinsulin ( Liu et al . , 2010b ) . Both preclinical and clinical data prove that in MIDY , it is the expression of misfolded proinsulin that triggers diabetes; yet MIDY is a rare disease . Of far broader significance is the β-cell failure that accompanies ‘garden variety’ T2D without INS mutations , and though the molecular pathogenesis of insulin deficiency in this condition remains murky ( Halban et al . , 2014 ) , β-cell ER stress is a recognized part of the disease . It has been suggested that β-cells compensate for insulin resistance by increasing insulin production that may eventually overwhelm the ER capacity for efficient protein folding , thereby provoking β-cell ER stress ( Back and Kaufman , 2012; Eizirik et al . , 2008; Herbert and Laybutt , 2016; Papa , 2012; Rabhi et al . , 2014; Volchuk and Ron , 2010 ) . However , in the absence of INS gene mutations , it has not been established the extent to which proinsulin misfolding is present in the early triggering stages of T2D , including prediabetes and mild dysglycemia — prior to more obvious islet failure including β-cell degranulation and dedifferentiation ( Accili et al . , 2016; Kahn , 1998; Kahn et al . , 2009 ) that occurs in both human islets ( Cinti et al . , 2016 ) and rodent islets ( Ishida et al . , 2017 ) . In this study , we have exploited several independent lines of evidence to establish the presence of aberrant disulfide-linked proinsulin complexes in the β-cells of human islets and model systems , in states that alter the ER folding environment , and in T2D progression prior to onset of β-cell dedifferentiation ( Bensellam et al . , 2018 ) or death ( Eizirik and Millard , 2014; Kanekura et al . , 2015; Marchetti et al . , 2012; Papa , 2012 ) .
Both murine islets and the INS1 ( rat ) pancreatic ß-cell line cells secrete successfully-folded proinsulin in addition to processed insulin . Native proinsulin folding requires formation of Cys ( B7 ) -Cys ( A7 ) , Cys ( B19 ) -Cys ( A20 ) and Cys ( A6 ) -Cys ( A11 ) disulfide pairs ( Haataja et al . , 2016 ) . One way to detect improperly folded wild-type proinsulin in pancreatic β-cells is to look for the possible presence of unpaired Cys residues . Alkylation of proinsulin Cys residues with 4-acetamido-4'-maleimidyl-stilbene-2 , 2'-disulfonate ( AMS ) adds 0 . 5 kD of molecular mass for each cysteine modified , shifting proinsulin from its normal molecular mass . As examined by immunoblotting with anti-proinsulin antibody , no modification by AMS could be detected in secreted recombinant human proinsulin or proinsulin from rodent islets , or INS cells ( e . g . , Figure 1—figure supplement 1A ) . Remarkably , however , alkylation of intracellular proinsulin with AMS in human islets caused a decrease in unmodified proinsulin accompanied by the appearance of proinsulin alkylated on at least one cysteine thiol ( Figure 1A ) . Alkylation of intracellular proinsulin was also observed in rodent islets ( Figure 1—figure supplement 1B ) . The presence of a free thiol in a significant subpopulation of proinsulin molecules can lead to inappropriate intermolecular disulfide attack on neighboring proinsulin molecules ( Cunningham et al . , 2017; Liu et al . , 2010a; Liu et al . , 2007; Wang et al . , 1999 ) . Antibodies have been described that recognize misfolded proinsulin molecules bearing intermolecular disulfide bonds ( Lee et al . , 2016; Wang et al . , 2011 ) . Indeed , WT proinsulin is predisposed to misfolding ( Haataja et al . , 2016 ) . Proinsulin misfolding has not been demonstrated to be exacerbated in ß-cells deficient for Ire1 or ATF6 , but it has been repeatedly found to be exacerbated in ß-cells with dysfunctional PERK ( caused either by gene knockout , dominant-negative mutant , or specific chemical inhibitor ) — leading to what has been described as the ‘proinsulin-impacted-ER’ phenotype ( Gupta et al . , 2010; Harding et al . , 2012; Scheuner et al . , 2005 ) . We performed immunoblotting of nonreducing SDS-PAGE samples with a monoclonal antibody that recognizes rodent proinsulin but not insulin ( mAb CCI-17 , see Materials and methods ) , with the intent to identify intermolecular disulfide-linked proinsulin complexes . Immunoblotting of either untreated ß-cells or those treated with vehicle alone detected proinsulin monomers and disulfide-linked dimers ( Figure 1B left ) the latter of which are , by definition , non-native . Moreover , whereas inhibition of PERK dramatically increased the intracellular accumulation of proinsulin and shifted its distribution to the classic impacted-ER phenotype ( Figure 1C ) ( Gupta et al . , 2010 ) , a remarkable ladder of higher molecular mass bands appeared upon Western blotting with mAb anti-proinsulin after nonreducing SDS-PAGE ( Figure 1B left ) . Despite this increase of intracellular proinsulin , secretion of proinsulin to the medium was not increased; furthermore , by nonreducing SDS-PAGE , secreted proinsulin was recovered exclusively as the monomeric form ( Figure 1B right ) , demonstrating efficient quality control in the secretory pathway . The intracellular accumulation of a ladder of proinsulin immunoreactive species upon nonreducing SDS-PAGE was not detected by conventional Western blotting with polyclonal anti-insulin , despite that this antibody identifies both proinsulin and proinsulin conversion intermediates ( Figure 1D left ) . However , dimers and higher order complexes were detected by Western blotting of the identical samples with mAb anti-proinsulin ( Figure 1D right ) , and all such higher bands , comprising the majority of intracellular proinsulin molecules , collapsed to monomers upon reduction of disulfide bonds ( Figure 1—figure supplement 1C ) . To confirm that the ladder of Western blotted bands detected by nonreducing SDS-PAGE specifically reflects disulfide-linked complexes of proinsulin , a similarly-generated sample was analyzed on a second-dimensional reducing SDS-PAGE , which demonstrated that nearly all bands in the nonreduced ladder contained proinsulin , with molecular masses expected of disulfide-linked complexes as simple as homodimers , homotrimers , homotetramers , and homopentamers , as well as higher order complexes ( Figure 1E ) . Quantitatively these complexes comprised 87% of all recovered proinsulin . This is very similar to what has been reported for the islets of Akita mice that express misfolded proinsulin from one mutant allele that entraps wild-type proinsulin expressed from three wild-type alleles ( Izumi et al . , 2003 ) . Indeed , Western blotting of nonreduced lysates of Akita mouse islets with anti-proinsulin demonstrated that the majority of proinsulin was recovered in aberrant disulfide-linked proinsulin complexes ( Figure 1F ) , which were converted to monomeric proinsulin upon SDS-PAGE under reducing conditions . To confirm that even in the absence of INS gene mutations , formation of disulfide-linked proinsulin complexes constitutes improper proinsulin folding , we exposed INS1 pancreatic beta cells to PERK inhibitor for times up to 20 hr . With increasing time of exposure , the majority of intracellular proinsulin accumulated in disulfide-linked complexes in the ß-cells ( Figure 2A second panel ) , and this increase was ultimately accompanied by decreased insulin content ( third panel ) as well as decreased proinsulin secretion ( final panel ) . These observations are consistent with ER quality control limiting the ER export of misfolded proinsulin resulting in diminished delivery to post-Golgi sites ( including the extracellular medium , also noted in Figure 1B ) . Significantly , treatment of intracellular proinsulin dimers and larger complexes with AMS , followed by nonreducing SDS-PAGE , revealed that each of the oligomeric species in the ladder of bands shifted up after alkylation , indicating that each of these forms still bears at least one unpaired cysteine thiol ( Figure 2B ) . These data provide a rationale for the propagation of disulfide-linked dimers into trimers , trimers into tetramers , etc . We found that pretreatment of cells with NEM before ( and during ) cell lysis results in the detection of dramatically less of the ladder of disulfide-linked proinsulin oligomers ( Figure 2C ) — and additionally , NEM pretreatment favors an increase in the detection of higher molecular weight proinsulin-containing complexes . Nevertheless , the relative abundance of the distinct disulfide-linked oligomeric species of proinsulin was essentially unchanged in cells that were either alkylated in situ with 20 mM N-ethyl maleimide prior to cell lysis , or not ( Figure 2C ) . These data render it unlikely that the detection of aberrant disulfide-linked complexes of nonmutant proinsulin is an artifact of cell lysis conditions . To explore whether human proinsulin ( Figure 1A ) is similarly predisposed to the aberrant disulfide-linked complex formation that was detected in rodent ß-cells , we first expressed recombinant human proinsulin in heterologous cells , followed by nonreducing SDS-PAGE and immunoblotting with a monoclonal antibody that reacts exclusively with the amino-terminal region of human C-peptide ( mAb 20G11 , see Materials and methods ) . Disulfide-linked proinsulin dimers along with a ladder of higher-order complexes already represented the majority of recombinant proinsulin species , and these forms accumulated to a dramatically higher level in cells treated overnight with PERK inhibitor ( Figure 2D ) . We then looked for the presence of such disulfide-linked dimers and higher order complexes formed by endogenous proinsulin in preparations of human islets from unrelated donors without a history of T2D . In several preparations , disulfide-linked dimers were prominent , along with a lesser abundance of higher-order complexes ( Figure 3A right panel ) . In human islets obtained from other individual donors , disulfide-linked dimers were less prominent ( Figure 2—figure supplement 1 and Figure 3—figure supplement 1A ) . Overnight treatment of human islets with PERK inhibitor resulted in a further increase in higher order disulfide-linked complexes ( Figure 3A right panel , and Figure 3—figure supplement 1A ) and an increase in total islet proinsulin as revealed by reducing SDS-PAGE ( Figure 3A left panel ) . These data indicate that aberrant disulfide-linked proinsulin dimers and higher complexes exist in human islets and their abundance responds to changes in environmental conditions within the ER . The immunodetection of both disulfide-linked dimers and higher-order complexes was blocked when pure human C-peptide competitor was included in the immunoblotting protocol ( Figure 3B ) , demonstrating specificity . PERK inhibitor has a rapid onset of action on its target , but it appears that the impact of this inhibition to globally alter the proinsulin folding environment of the ER ( Wang et al . , 2013 ) may take a half-day or more ( Figure 2A ) . More immediately , nascent proinsulin binds the hsp70 family member , BiP ( Liu et al . , 2005; Scheuner et al . , 2005 ) ; thus , for a more direct perturbation , we exposed islets to the bacterial SubAB protease that is endocytosed and retrieved to the ER lumen where it cleaves the ER chaperone BiP within a matter of a few hours or less ( Paton et al . , 2006 ) . In mouse islets , BiP was ≥90% destroyed within 2 hr after SubAB addition ( Figure 3C upper left ) , and immunoblotting revealed that this treatment shifted the majority of proinsulin into larger disulfide-linked complexes ( Figure 3C lower right ) . Similarly , in a rodent ß-cell line , improperly folded proinsulin began to migrate increasingly in disulfide-linked complexes 80 min after SubAB addition when only a portion of intracellular BiP had been destroyed ( Figure 3—figure supplement 1B ) . BiP cleavage was also confirmed by appearance of the ~28 kD C-terminal BiP cleavage fragment ( Figure 3—figure supplement 1C ) . In the islets of humans not known to have T2D , improperly folded disulfide-linked proinsulin dimers were already apparent prior to SubAB addition , and an increase in larger-sized covalent proinsulin complexes was apparent at 4 hr of SubAB treatment ( Figure 3D lower panel ) . These data demonstrate an acute increase in improper folding of human proinsulin , without any INS gene mutation , under conditions of BiP deficiency within the ß-cell ER luminal environment . The hsp90 family member of the ER , GRP94 , also impacts on proinsulin handling in ß-cells , especially resulting in aberrant post-ER processing with markedly abnormal appearing secretory granules ( Ghiasi et al . , 2019 ) ; however , treatment of ß-cells with GRP94 inhibitor ( PU-WS13 , 20 µM ) even for 24 hr did not promote proinsulin disulfide-linked complex formation , and did not exacerbate the proinsulin disulfide-linked complex formation that was triggered by an acute ( 3 hr ) loss of BiP ( Figure 4A ) . Immunofluorescence microscopy demonstrated that after SubAB treatment of ß-cells , the intracellular distribution of proinsulin shifted from its usual predominant juxtanuclear localization [in the Golgi region from which newly-made insulin granules emerge ( Haataja et al . , 2013 ) to a co-localization with the ER marker , calnexin ( Figure 4B ) . Altogether , these data strongly indicate that proinsulin disulfide-linked complexes are misfolded and are retained within the ER compartment . The predominant juxtanuclear Golgi-regional distribution of proinsulin is a feature of normal rodent islets ( Orci et al . , 1985 ) ( Figure 4C first set of panels ) . Within the first 4–5 months of life , leptin receptor mutant LepRdb/db mice in a C57BL/6 background become severely diabetic and develop a paucity of islet ß-cells immunostainable for mature insulin — indeed , the fraction of islet ß-cells that exhibit little or no insulin immunostaining is known to increase by more than 5-fold compared to the islets of age-matched control mice ( Ishida et al . , 2017 ) . However , we observed that proinsulin-positive immunostaining amongst the cells in these diabetic islets was more widespread and the intracellular distribution of proinsulin within these islet ß-cells was no longer as concentrated in the Golgi region ( Figure 4C second and third set of panels ) with increased localization in the ER ( Figure 4—figure supplement 1 ) . It has been established that limiting food intake of these animals can substantially increase the percentage of strongly insulin-immunopositive ß-cells per islet ( Ishida et al . , 2017 ) . Indeed , upon fasting overnight , the islets of diabetic LepRdb/db mice exhibited a more robust immunostaining of mature insulin ( Figure 4C fourth and fifth set of panels ) . Additionally , after fasting , there was a shift in intracellular proinsulin back towards a juxtanuclear distribution ( Figure 4C fourth and fifth set of panels ) , consistent with previous reports ( Alarcon et al . , 2016 ) . The data of Figure 4 strongly suggest that in ad lib fed LepRdb/db diabetic mice , initial insulin depletion does not preclude ongoing proinsulin expression; moreover , a shift of proinsulin distribution towards an ER-like pattern ( Figure 4—figure supplement 1 ) may suggest a stressed state that may be preventable and to some extent reversible by limiting ß-cell secretory stimulation ( i . e . , ‘ß-cell rest’ ) ( Ishida et al . , 2017 ) . We wondered if the ER-like distribution of intracellular proinsulin in LepRdb/db mice might be explained by ( nonmutant ) proinsulin folding exceeding the ER folding capacity of these ß-cells , resulting in the accumulation of improperly folded species . With this in mind , we performed proinsulin immunoblotting after nonreducing and reducing SDS-PAGE in LepRdb/db islet lysates , with islet lysates from wild-type mice , or those bearing only one of 4 functional Ins alleles , as controls . Under reducing conditions , >99% of proinsulin was monomeric ( and insulin was recovered as the reduced insulin B-chain , Figure 4D right ) . We examined proinsulin in the islet lysates of 5 additional prediabetic LepRdb/db mice — all were similar to the islets of two animals shown in Figures 4D ( 4 and 6 weeks of age with random blood glucose levels of 101 and 118 mg/dL , respectively ) containing a large steady state level of proinsulin ( seen upon Western blotting of reducing SDS-PAGE ) with >90% of molecules entangled in disulfide-linked dimers and higher-order complexes ( seen by Western blotting upon nonreducing SDS-PAGE , Figure 4D ) . Accumulation of misfolded proinsulin in the ER of LepRdb/db ß-cells was always present before the onset of diabetes . These bands are indeed proinsulin , as they were absent from islets of mice depleted of three of four Ins alleles; Figure 4D lane 3 ) . Importantly , improperly folded proinsulin was also detectable in WT mouse islets , albeit at a much lower abundance ( Figure 4D lane 4 ) . Because leptin receptors might have direct actions on ß-cells ( Marroquí et al . , 2012 ) , we also examined islets from LepR-Nkx2 . 1 KO mice that are deficient for leptin receptor in the hypothalamus but not in the islets ( Ring and Zeltser , 2010 ) . Here too , pancreatic islets from these hyperphagic , obese , glucose intolerant animals demonstrated markedly increased abundance of disulfide-linked proinsulin complexes with >90% of proinsulin entangled in such complexes ( Figure 4E ) . These data indicate that an increase in improper proinsulin folding in leptin receptor-deficient animals is secondary to a prediabetic state and unrelated to leptin receptor function in ß-cells . Crucially , these data also demonstrate that improper proinsulin folding is already detectable at a time that has been associated with ER stress response ( Herbert and Laybutt , 2016 ) but before onset of chronic hyperglycemia associated with pancreatic insulin deficiency that has been attributed to a loss of functional ß-cell mass [regardless of whether this is due to de-differentiation ( Bensellam et al . , 2018 ) or ß-cell death ( Eizirik and Millard , 2014; Kanekura et al . , 2015; Marchetti et al . , 2012; Papa , 2012 ) or both] . Based on the work of Laybutt et al . ( 2007 ) , we examined islet protein content of p58ipk ( encoded by DNAJC3 ) as a reliable marker of islet ER stress response , and found that the levels of this ER co-chaperone of BiP began to increase in ad lib fed LepRdb/db mice even before the animals have reached a random blood glucose of 160 mg/dL ( Figure 5A ) . Islet p58ipk protein levels also tended to be elevated in LepR-Nkx2 . 1 KO mice ( Figure 5B ) . In the islets of young , normoglycemic homozygous LepRdb/db male mice that are destined for diabetes , proinsulin disulfide-linked complexes ( Figure 5C upper left panel ) were increased slightly above the level observed in heterozygous LepRdb/+ males ( that do not progress to diabetes ) , and islet insulin levels were similarly increased ( lower left panels ) . With a random blood glucose level in the mid 200’s ( mg/dL ) , islet proinsulin was notably increased ( Figure 5C upper second panel ) and comprised >90% of disulfide-linked proinsulin complexes ( upper first panel ) whereas simultaneously , islet insulin levels were dramatically decreased ( lower left panels ) . With a random blood glucose level >500 mg/dL , islet proinsulin and insulin levels were both severely diminished ( Figure 5C left panels ) . In female LepRdb/db mice with a random blood glucose ranging from 156 to 294 mg/dL , insulin levels declined compared to the LepRdb/+ control ( Figure 5C lower right panels ) . However , for females in this glycemic range , as in males , islet proinsulin was increased ( Figure 5 upper right panel ) and once again , >90% was entangled in disulfide-linked proinsulin complexes ( Figure 5 upper third panel ) . Additionally , with a random blood glucose of 455 mg/dL , both proinsulin and insulin were severely diminished ( Figure 5C last two sets of panels ) indicating islet decompensation . During this progression to diabetes , intracellular proinsulin distribution that initially displayed a prominent juxtanuclear pattern with good co-localization to the Golgi region ( GM130 Golgi marker ) tended to shift towards a staining pattern that was more spread in the cytoplasm , with increased co-localization with the ER marker , calnexin ( Figure 5—figure supplement 1 ) ; although even in the fully diabetic state there appeared to be increased juxtanuclear ( Golgi-like ) proinsulin distribution after overnight fasting ( Figure 5—figure supplement 1 ) . Cys ( A20 ) and Cys ( B19 ) are two of the three most reactive thiols of proinsulin , and they initiate covalent association of B- and A-domains that are required for proinsulin export from the ER ( Haataja et al . , 2016 ) . Nevertheless , in the redox environment of the pancreatic ß-cell ER , a human mutant proinsulin that retains only two cysteines , named ‘keep-B19/A20’ cannot undergo efficient intramolecular oxidation ( Haataja et al . , 2016 ) . Reactive free thiol availability ( Figure 1A ) is a key to proinsulin participation in intermolecular disulfide-linked complex formation . Indeed , each of the myc-tagged human proinsulins keep-B7/A7 and keep-A6/A11 , in addition to keep-B19/A20 , exhibited free thiol availability as determined by alkylation with AMS ( Figure 6—figure supplement 1 ) . We expressed each of these recombinant proinsulin mutants in the INS-832/13 ß-cell line ( which already expresses endogenous proinsulin ) . Because these two-cysteine mutant human proinsulins cannot be exported from the ER and are excellent ERAD substrates in pancreatic ß-cells ( Haataja et al . , 2016 ) , these molecules were largely undetected by immunoblotting . However , in ß-cells treated with proteasome inhibitor ( MG132 for 7 hr ) , myc-tagged proinsulin monomers were detected in all cases ( Figure 6A ) . Remarkably , not only were these constructs capable of forming disulfide-linked proinsulin homodimers , but the keep-B19/A20 construct in particular recapitulated the situation in which the majority of molecules migrated as disulfide-linked proinsulin complexes ( Figure 6A ) . With or without MG132 , this behavior was also observed upon expression in 293 T cells that lack endogenous proinsulin ( Figure 6B ) , and these complexes appeared quite similar to those observed in human islets ( Figure 3A , B ) . To determine if Cys ( B19 ) /Cys ( A20 ) are both necessary and sufficient for generating the full ladder of disulfide-linked proinsulin complexes , we compared the three different human mutant proinsulins noted above that each retain only one set of potential disulfide partners , and a human proinsulin mutant named ‘lose-B19/A20’ in which Cys ( B19 ) and Cys ( A20 ) are mutated whereas positions B7 , A7 , A11 and A20 remain as cysteines . Once again , of the mutant human proinsulins that retain only one set of potential disulfide partners , only keep-B19/A20 could efficiently recreate the entire ladder of disulfide-linked complexes similar to that seen for wildtype proinsulin , irrespective of the presence or absence of an engineered myc epitope tag in the C-peptide ( Figure 6C left panel ) . However , lose-B19/A20 did not efficiently recreate the ladder of disulfide-linked complexes despite entrapment in the ER in a proinsulin construct bearing four Cys residues . These data indicate that the Cys ( B19 ) /Cys ( A20 ) pair is both necessary and sufficient for efficient propagation of disulfide-linked complexes of improperly-folded proinsulin . In these experiments , we were struck by the observations 1 ) that all 2-Cys proinsulin mutants have the capability to at least weakly form a covalent dimer , which is essential to the further propagation of these improperly folded complexes into trimers , tetramers , and higher order complexes ( Figure 2B ) , and 2 ) a small fraction of proinsulin dimers appeared to remain even after reducing SDS-PAGE ( Figure 4D; Figure 1—figure supplement 1C ) . To understand these behaviors , we expressed a series of mutant human proinsulins that retain only a single cysteine ( and thus cannot make more than one intermolecular disulfide bond ) . Although five of the six Cys residues could make a homotypic covalent association , Cys ( A7 ) preferred not to make the disulfide bond ( Figure 7A left panel; quantified in Figure 7B ) . More significantly , Cys ( B19 ) not only covalently homodimerized with exuberance ( Figure 7A left panel ) but this Cys ( B19 ) -Cys ( B19 ) bond could not be broken at room temperature in SDS-gel sample buffer even in the presence of 200 mM dithiothreitol ( Figure 7A right panel ) . These data indicate that a Cys ( B19 ) -Cys ( B19 ) disulfide bond between proinsulin monomers predisposes to strongly-associated covalent complexes that are automatically misfolded by omitting the crucial intramolecular Cys ( B19 ) -Cys ( A20 ) disulfide bond , which leaves the critical reactive Cys ( A20 ) residue unpaired and available for further disulfide infidelity . Such an initiating event can thus form a nidus for assembly of larger misfolded disulfide-linked proinsulin complexes ( Figure 7—figure supplement 1B ) .
It is recognized from human genome-wide association studies and animal models that progression from insulin resistance to pancreatic ß-cell dysfunction is a linchpin in the development of T2D . Pancreatic ß-cell ER stress is one of the most frequently described components of T2D in humans and animal models ( Back and Kaufman , 2012; Berry et al . , 2018; Cnop et al . , 2017; Laybutt et al . , 2007; Marchetti et al . , 2007; Rabhi et al . , 2014; Yang et al . , 2016 ) . While protein misfolding caused by mutations ( in so-called conformational diseases ) is one recognized cause of ER stress , the proximal trigger of beta cell ER stress early in the progression of T2D is unknown , but has been the subject of much speculation . It is indisputable that ß-cell ER stress can be triggered by proinsulin misfolding in the setting of INS gene coding sequence mutations ( Liu et al . , 2018 ) but there are also strong reasons to think that even in the absence of INS gene mutations , proinsulin misfolding could be an early feature in the progression of T2D ( Arunagiri et al . , 2018; Riahi et al . , 2018; Scheuner and Kaufman , 2008 ) . In the present study , we have exploited several independent lines of experimentation to identify a significantly increased population of improperly folded proinsulin in the ER of prediabetic and diabetic ß-cells . We establish that proinsulin misfolding includes the inability to successfully complete its three internal disulfide bonds , with a subfraction of proinsulin molecules in the ER bearing unpaired cysteine residues . This population of improperly folded proinsulin molecules enters into aberrant disulfide-linked partnerships with other proinsulin molecules in the ER , resulting in misfolded proinsulin complexes that are for the first time identified in the islets of human beings . There is strong reason to believe that proinsulin disulfide-linked complex formation provides a status report on the health of the ß-cell ER folding environment . Many studies point to the idea that the activity of ER stress ( UPR ) sensor proteins are required to actively maintain a proper ER chaperone and oxidoreductase environment for optimal proinsulin folding ( Harding et al . , 2012; Hassler et al . , 2015; Sowers et al . , 2018; Tsuchiya et al . , 2018 ) . Recent work indicates that protein disulfide isomerase is one of the ER luminal factors that regulates the balance of proinsulin disulfide-linked complexes and monomers ( Jang et al . , 2019 ) . Additionally , BiP plays a central role in ER stress sensing ( Amin-Wetzel et al . , 2017 ) . We show here that even a modest decrease of BiP levels results in improperly folded proinsulin rapidly accumulating in disulfide-linked complexes ( Figure 3—figure supplement 1B ) , supporting that proinsulin folding is highly sensitive to changes in the ER folding environment , which can include excessive proinsulin biosynthesis , altered chaperone and/or oxidoreductase expression , and changes in the rate of clearance of misfolded proinsulin molecules ( Arunagiri et al . , 2018; Xu et al . , 2018 ) . We believe that these and other ER luminal factors are needed to enhance the efficiency of formation of the proinsulin intramolecular Cys ( B19 ) -Cys ( A20 ) disulfide bond . Not only is this internal disulfide critical to additional native proinsulin disulfide pairing ( Haataja et al . , 2016 ) ( Figure 7—figure supplement 1A ) , but the availability of free Cys ( B19 ) and Cys ( A20 ) can recapitulate the entire ladder of improperly disulfide linked proinsulin complexes . Several pathways of disulfide propagation are possible , highlighted in Figure 7—figure supplement 1B . However , those complexes bearing a homotypic Cys ( B19 ) -Cys ( B19 ) covalent bond are likely to be the most difficult to successfully isomerize , as this bond cannot be broken in vitro even in the presence of SDS plus 200 mM DTT ( Figure 7A ) . A specific goal of T2D research is to better understand the natural history of ß-cell failure ( Halban et al . , 2014 ) . What has recently emerged using the LepRdb/db mouse model , is that beginning around 4–5 months of life , after hyperglycemia has appeared , ß-cells appear to be in the early stages of dedifferentiation ( Ishida et al . , 2017 ) , and with a similar time course , ß-cell apoptosis may occur in parallel ( Wu et al . , 2015 ) . However , clearly there are important islet changes that occur considerably before these events ( Do et al . , 2016 ) including ß-cell ER stress response activation ( Figure 5A , B ) ( Herbert and Laybutt , 2016 ) . Figure 8 presents a schematic describing our hypothesis regarding how proinsulin misfolding fits into the paradigm of what is known about T2D progression in the LepRdb/db model . Our work highlights that throughout the first , compensated stage , improperly folded proinsulin is increased but islet insulin content is still robust ( Figures 4D and 5C ) . In the second , decompensating stage , islet proinsulin levels are actually further increased , but much of this is improperly folded proinsulin in the ER , and at this stage , islet insulin levels begin to drop ( Figure 5 ) . It is at a still higher level of hyperglycemia that both proinsulin and insulin steady state levels are low ( Figure 8 ) , which correlates with the time when ß-cell dedifferentiation and ß-cell death have been reported . It should be noted that even at this time , the low-level proinsulin protein that is still expressed is recovered >90% in aberrant disulfide linked complexes , and even partial restoration of secretory pathway function requires ß-cell rest ( Alarcon et al . , 2016 ) . We suspect that throughout the progression of ß-cell stress in T2D , quality control of proinsulin anterograde export from the ER is nevertheless maintained . We note that we have been unable to detect secreted proinsulin that can be alkylated with AMS , or that is recovered in disulfide-linked complexes , even from islets or cells in which proinsulin has accumulated intracellularly in disulfide-linked complexes at massive levels . However , experiments such as these will need to be rigorously tested with islets from human T2D subjects , as these patients are known to have abnormally high levels of immunoreactive proinsulin in the circulation ( Porte and Kahn , 2001 ) . In conclusion , our data establish the presence of a previously underappreciated population of aberrant proinsulin complexes that accumulates in prediabetic conditions and persists until ß-cell failure ensues . We note that the proinsulin complexes defined by nonreducing SDS-PAGE highlight a core of covalently-associated proteins; further work will be needed to explore additional protein partners including those that may be noncovalently associated . Nevertheless , the data presented herein establish disulfide-linked complexes of proinsulin as one of the earliest tissue biomarkers indicating ß-cell secretory pathway dysfunction , which is associated with ER stress and ultimate insulin deficiency that occurs in the natural progression of T2D .
AMS , DTT , N-ethylmaleimide , PERK inhibitor ( GSK2656157 ) , MG132 , Cycloheximide , and all other chemicals were purchased from Sigma-Aldrich or ThermoFisher Scientific . SDS-PAGE 4–12% Bis-Tris or 4–20% Tris-Glycine NuPage gels were obtained from ThermoFisher . Antibodies used in the present work are as follows: The mutant LepRdb allele was carried in the C57BLKS/J background; heterozygotes were cross-bred to generate LepRdb/db homozygotes . Wild-type and Akita mutant mice were maintained in the C57BL/6J background . All of the preceding breeder mice were obtained from JAX . Ins1−/− , Ins2+/− mice generated in a C57BL/6 background were intercrossed as previously described ( Duvillie et al . , 1997; Duvillié et al . , 2002 ) . In this study we restricted our analyses to males because Akita diabetic males develop progressive hyperglycemia to which the females are resistant ( Yoshioka et al . , 1997 ) . LepRNkx2 . 1 KO mice were identical to those previously described ( Ring and Zeltser , 2010 ) . Mice were euthanized by CO2 narcosis as per an approved institutional animal protocol . The pancreas was rapidly excised , minced in ice-cold PBS , and digested in 4 mL of Collagenase P ( Roche ) 1 . 5 mg/mL in Hank’s Balanced Salt Solution containing calcium and magnesium , in a shaking water bath for 30 min at 37 °C . The digestion was terminated in 40 mL ice-cold PBS ( -Ca/-Mg ) and the digested tissue washed twice in this buffer . The sedimented tissue digest was then overlaid with 3 mL Histopaque-1077 and further overlayed with 6 mL ice cold PBS ( -Ca/-Mg ) before centrifugation ( 3000 rpm for 20 min at 10 °C with no brake ) and transfer of the mid-layer of the Histopaque gradient to a 15 ml tube for two further washes in ice-cold PBS . Finally , the sedimented tissue was transferred to a petri dish in ice-cold RPMI- 1640 buffer and islets hand-picked to purity . In experiments involving drug or compound treatment , the islets were finally recovered overnight in RPMI-1640 supplemented with 10% fetal bovine serum ( FBS ) , 10 mM HEPES pH 7 . 35 , 1 mM sodium pyruvate and 0 . 05 mM beta-mercaptoethanol , in a humidified 5% CO2 incubator at 37 °C . For some experiments , islets were snap frozen in liquid nitrogen and stored frozen prior to analysis . Human islets were obtained either from Prodo Labs , or from the NIDDK-funded Integrated Islet Distribution Program ( IIDP; NIH UC4-DK098085 ) and maintained in a humidified 5% CO2 incubator at 37°C . Human islets were cultured ex vivo for up to 96 hr in Prodo PIM ( R ) islet-specific tissue culture medium supplemented with 10% FBS and Prodo PIM ( G ) glutamine/glutathione supplement , plus penicillin/streptomycin . Rat pancreatic beta cell lines INS1E and INS-832/13 were cultured in RPMI-1640 medium supplemented with 10% FBS , 10 mM HEPES pH 7 . 35 , 1 mM sodium pyruvate , penicillin/streptomycin and 0 . 05 mM beta-mercaptoethanol . HEK293T human cells lines were cultured in DMEM supplemented with 10% FBS and penicillin/streptomycin . INS1E cell or mouse or human islet lysate , each diluted in reaction buffer ( 50 mM Tris pH 7 . 4 , 1% SDS final concentrations ) were heated to 95 °C for 5 min , cooled to room temperature and then incubated further in the same buffer containing 4-Acetamido-4'-Maleimidylstilbene-2 , 2'-Disulfonic Acid , Disodium Salt ( AMS , ThermoFisher ) for 1 hr at 37°C . In one experiment , 2 mM DTT was added during the initial boiling step prior to aklylation . No differences in alkylation were observed at AMS doses ranging from 6 mM to 20 mM . Non-alkylated controls underwent all the same incubations , in the same buffers , in parallel . The INS1E cells bathing media were also similarly tested for AMS-reactive proinsulin species . AMS-treated and untreated controls were analyzed by nonreducing or reducing SDS-PAGE and immunoblotting as described below . The generation of myc-tagged keep-B7/A7 , keep-B19/A20 , and keep-A6/A11 were described previously ( Haataja et al . , 2016 ) . These plasmids were used as templates for further mutagenesis to create six single-cysteine myc-tagged proinsulin mutants using the QuikChange site-directed mutagenesis kit ( Agilent ) . All resulting plasmids encoding corresponding proinsulin mutations were confirmed by direct DNA sequencing . The expression plasmid encoding hPro-CpepMyc has been previously described ( Haataja et al . , 2013 ) . 293 T cells , INS1E cells , and INS-832/13 cells at 70–80% confluency were transiently transfected using Lipofectamine 2000 ( ThermoFisher ) as per the manufacturer’s instructions . A medium change was performed 5 hr post-transfection and the cells were lysed at 36 or 48 hr . After removal of media , cells were washed once with ice-cold PBS and lysed in RIPA buffer ( 10 mM Tris pH 7 . 4 , 150 mM NaCl , 0 . 1% SDS , 1% NP40 , 2 mM EDTA ) plus protease inhibitor/phosphatase inhibitor cocktail ( Sigma-Aldrich ) or directly in Laemmli gel sample buffer . Lysates in RIPA buffer were immediately spun at 10 , 000 rpm for 10 min at 4 °C and the supernatants analyzed further or stored at −80°C . Islets that had been quick frozen were placed on ice and RIPA buffer containing protease inhibitor cocktail and phosphatase inhibitor was added . Lysis was carried out by gently pipetting or by syringe through a 30G needle . Total protein concentration in the lysate was determined by BCA or Bramhall assay , and 5–10 μg of samples prepared in SDS sample were resolved by SDS-PAGE in 4–12% Bis-Tris NuPAGE gels ( Invitrogen ) at 200 V for 30 min . Nonreducing gels were incubated in a solution containing 25 mM dithiothreitol ( DTT ) for 10 min at room temperature prior to electrotransfer . A lane excised from the nonreducing slab gel loaded with lysate from INS1E cells treated with PERK inhibitor , was incubated for 15 min at room temperature in Tris-Glycine pH 6 . 8 plus 20 mM DTT . The lane was then laid horizontally on top of a 10% SDS-polyacrylamide resolving gel and sealed in place in stacking gel . A single-tooth comb was introduced at one end of the stacking gel to introduce a one-dimensional reduced sample of proinsulin containing cell lysate ( which runs as proinsulin monomer ) . The gel was run at 200 V for 45 mins in Tris-Glycine buffer pH 8 . 8 followed by electrotransfer . Transfer membranes were rinsed once in TBST ( 15 mM Tris , 150 mM NaCl , 0 . 1% Tween-20 ) , blocked using TST plus 5% BSA for 1 hr at room temperature , and then washed four times ( 5 min each ) in TBST . Primary antibody and secondary antibodies were diluted in TBST and each incubation was 1 hr at room temperature followed by four washes in TBST . On occasion , primary antibodies were incubated overnight at 4 °C . Secondary antibodies were all HRP-conjugates . Development of immunoblots used enhanced chemiluminescence ( Immobilon , Millipore , or SuperSignal West Pico PLUS , Thermo Fisher Scientific ) with images captured using a Fotodyne gel imager . Pancreatic paraffin sections were de-paraffinized with Citrisolv ( Fisher Scientific ) for 5 min at room temperature , re-hydrated in a series of 8 min ethanol incubations: 100% , 95% , 70% , and distilled water . Antigen retrieval was carried out using Retrieve-ALL 1 Universal pH 8 ( BioLegend ) and slides were heated in a microwave , cooled for 30 min at room temperature , and incubated once with PBS for 5 min . Blocking was performed in 150 μL blocking buffer ( 3% BSA prepared in TBS and 0 . 2% Triton X-100 ) per section for 2 hr at room temperature . Blocking buffer was removed and 150 μL per section of primary antibody appropriately diluted in TBS plus 3% BSA and 0 . 2% Tween-20 was incubated overnight at 4 °C . The primary antibody was removed and the slide washed twice with TBS/0 . 1% Tween-20 . Then , 150 μL secondary antibody ( 1:500 dilution , prepared in antibody buffer ) per section was incubated for 1 hr at room temperature . Slides were washed thrice with TBS/0 . 1% Tween-20 , mounted with a drop of prolong gold anti-fade reagent with DAPI ( ThermoFisher ) and a cover slip affixed . For immunocytochemistry , INS1E cells were grown in an 8-well Millicell EZ SLIDE ( Millipore-SIGMA ) . Cells were allowed to reach 60–80% confluency before addition of PERK inhibitor ( PERKi ) or vehicle . After 18 hr of PERKi treatment , the medium was removed and the cells were fixed in 3 . 7% formaldehyde in PBS pH 7 . 4 , for 20 min at room temperature , rinsed once with PBS , and permeabilized with 0 . 4% Triton X-100 in TBS for 20 min at room temperature . The cells were washed thrice with TBS and then incubated in blocking buffer as described above . Thereafter samples were incubated overnight at 4°C with 100 μL of appropriately diluted primary antibody in 3% BSA/TBS/0 . 2% Tween . The cells received four 15 min washes in TBS , and then incubated with secondary antibody ( 1:500 dilution ) for 1 hr at room temperature . The cells were then washed thrice with TBS and finally mounted with a drop of Prolong Gold anti-fade reagent containing DAPI ( ThermoFisher ) and a cover slip affixed and the slide incubated in the dark for 24 hr at room temperature . A similar immunocytochemistry protocol was followed for INS-832/13 cells after SubAB treatment ( 4 hr ) .
Biosynthesis of insulin – critical to metabolic homeostasis – begins with folding of the proinsulin precursor , including formation of three evolutionarily conserved intramolecular disulfide bonds . Remarkably , normal pancreatic islets contain a subset of proinsulin molecules bearing at least one free cysteine thiol . In human ( or rodent ) islets with a perturbed endoplasmic reticulum folding environment , non-native proinsulin enters intermolecular disulfide-linked complexes . In genetically obese mice with otherwise wild-type islets , disulfide-linked complexes of proinsulin are more abundant , and leptin receptor-deficient mice , the further increase of such complexes tracks with the onset of islet insulin deficiency and diabetes . Proinsulin-Cys ( B19 ) and Cys ( A20 ) are necessary and sufficient for the formation of proinsulin disulfide-linked complexes; indeed , proinsulin Cys ( B19 ) -Cys ( B19 ) covalent homodimers resist reductive dissociation , highlighting a structural basis for aberrant proinsulin complex formation . We conclude that increased proinsulin misfolding via disulfide-linked complexes is an early event associated with prediabetes that worsens with ß-cell dysfunction in type two diabetes .
Our body fine-tunes the amount of sugar in our blood thanks to specialized ‘beta cells’ in the pancreas , which can release a hormone called insulin . To produce insulin , the beta cells first need to build an early version of the molecule – known as proinsulin – inside a cellular compartment called the endoplasmic reticulum . This process involves the formation of internal staples that keep the molecule of proinsulin folded correctly . Individuals developing type 2 diabetes have spikes of sugar in their blood , and so their bodies often respond by trying to make large amounts of insulin . After a while , the beta cells can fail to keep up , which brings on the full-blown disease . However , scientists have discovered that early in type 2 diabetes , the endoplasmic reticulum of beta cells can already show signs of stress; yet , the exact causes of this early damage are still unknown . To investigate this , Arunagiri et al . looked into whether proinsulin folds correctly during the earliest stages of type 2 diabetes . Biochemical experiments showed that even healthy beta cells contained some misfolded proinsulin molecules , where the molecular staples that should fold proinsulin internally were instead abnormally linking proinsulin molecules together . Further work revealed that the misfolded proinsulin was accumulating inside the endoplasmic reticulum . Finally , obese mice that were in the earliest stages of type 2 diabetes had the highest levels of abnormal proinsulin in their beta cells . Overall , the work by Arunagiri et al . suggests that large amounts of proinsulin molecules stapling themselves to each other in the endoplasmic reticulum of beta cells could be an early hallmark of the disease , and could make it get worse . A separate study by Jang et al . also shows that a protein that limits the misfolding of proinsulin is key to maintain successful insulin production in animals eating a Western-style , high fat diet . Hundreds of millions of people around the world have type 2 diabetes , and this number is rising quickly . Detecting and then fixing early problems associated with the condition may help to stop the disease in its track .