Characterization of proinsulin- and proglucagon-converting activities in isolated islet secretory granules

The conversion of proglucagon and proinsulin by secretory granules isolated from both prelabeled and unlabeled anglerfish islets was investigated. Either granules isolated from tissue labeled with [3H]tryptophan and [14C]isoleucine or [35S]cysteine, or lysed granules from unlabeled tissue to which exogenously labeled prohormones had been added were incubated under various conditions. Acetic acid extracts of these granule preparations were analyzed for prohormone and hormone content by gel filtration. Both prelabeled and lysed, unlabeled secretory granules converted radiolabeled precursor peptides (Mr 8,000- 15,000) to labeled insulin and glucagon. The accuracy of the cleavage process was established by demonstrating comigration of products obtained from in vitro cleavage with insulin and glucagon extracted from intact islets using electrophoresis and high-pressure liquid chromatography (HPLC). The pH optimum for granule-mediated conversion was found to be in the range of pH 4.5-5.5. Conversion of both proglucagon and proinsulin by secretory granules was significantly inhibited in the presence of antipain, leupeptin, p- chloromercuribenzoate (PCMB) or dithiodipyridine (DDP) but not chloroquine, diisopropyl fluorophosphate, EDTA, p-nitrophenyl guanidinobenzoate, soybean trypsin inhibitor, or N-p-tosyl-L-lysine chloromethyl ketone HCl. The inhibitory action of PCMB and DDP was reversed in the presence of dithiothreitol. Both membranous and soluble components of the secretory granules possessed significant converting activity. HPLC and electrophoretic analysis of cleavage products demonstrated that the converting activities of the membranous and soluble components were indistinguishable. The amount of inhibition of proinsulin and proglucagon conversion caused by 600 micrograms/ml porcine proinsulin was significantly lower than that caused by the same concentration of unlabeled anglerfish precursor peptides. These results indicate that the proinsulin and proglucagon converting enzyme(s) in the anglerfish pancreatic islet is a unique intracellular thiol proteinase(s) that may be granule membrane-associated and may require the presence of prohormone sequences in addition to the dibasic residues at cleavage sites for substrate recognition and/or binding.     

Processing of synthetic pro-islet amyloid polypeptide (proIAPP) 'amylin' by recombinant prohormone convertase enzymes, PC2 and PC3, in vitro.

Islet amyloid polypeptide (IAPP), amylin, is the constituent peptide of pancreatic islet amyloid deposits which form in islets of Type 2 diabetic subjects. Human IAPP is synthesized as a 67-residue propeptide in islet beta-cells and colocalized with insulin in beta-cell granules. The mature 37-amino acid peptide is produced by proteolysis at pairs of basic residues at the C- and N-termini of the mature peptide. To determine the enzymes responsible for proteolysis and their activity at the potential cleavage sites, synthetic human proIAPP was incubated (0.5-16 h) with recombinant prohormone convertases, PC2 or PC3 at appropriate conditions of calcium and pH. The products were analysed by MS and HPLC. Proinsulin was used as a control and was cleaved by both recombinant enzymes resulting in intermediates. PC3 was active initially at the N-terminal-IAPP junction and later at the C-terminus, whereas initial PC2 activity was at the IAPP-C-terminal junction. Processing at the basic residues within the C-terminal flanking peptide rarely occurred. There was no evidence for substantial competition for the processing enzymes when the combined substrates proinsulin and proIAPP were incubated with both PC2 and PC3. As proinsulin cleavage is sequential in vivo (PC3 active at the B-chain-C-peptide junction, followed by PC2 at A chain-C-peptide junction), these data suggest that proteolysis of proIAPP and proinsulin is coincident in secretory granules and increased proinsulin secretion in diabetes could be accompanied by increased production of proIAPP.     

Newly synthesized proinsulin/insulin and stored insulin are released from pancreatic B cells predominantly via a regulated, rather than a constitutive, pathway

The pancreatic B cell has been used as a model to compare the release of newly synthesized prohormone/hormone with that of stored hormone. Secretion of newly synthesized proinsulin/insulin (labeled with [3H]leucine during a 5-min pulse) and stored total immunoreactive insulin was monitored from isolated rat pancreatic islets at basal and stimulatory glucose concentrations over 180 min. By 180 min, 15% of the islet content of stored insulin was released at 16.7 mM glucose compared with 2% at 2.8 mM glucose. After a 30-min lag period, release of newly synthesized (labeled) proinsulin and insulin was detected; from 60 min onwards this release was stimulated up to 11-fold by 16.7 mM glucose. At 180 min, 60% of the initial islet content of labeled proinsulin was released at 16.7 mM glucose and 6% at 2.8 mM glucose. Specific radioactivity of the released newly synthesized hormone relative to that of material in islets indicated its preferential release. A similar degree of isotopic enrichment of released, labeled products was observed at both glucose concentrations. Quantitative HPLC analysis of labeled products indicated that glucose had no effect on intracellular proinsulin to insulin conversion; release of both newly synthesized proinsulin and insulin was sensitive to glucose stimulation; 90% of the newly synthesized hormone was released as insulin; and only 0.5% of proinsulin was rapidly released (between 30 and 60 min) in a glucose-independent fashion. It is thus concluded that the major portion of released hormone, whether old or new, processed or unprocessed, is directed through the regulated pathway, and therefore the small (less than 1%) amount released via a constitutive pathway cannot explain the preferential release of newly formed products from the B cell.     

Stimulation by ATP of proinsulin to insulin conversion in isolated rat pancreatic islet secretory granules. Association with the ATP-dependent proton pump

Isolated rat pancreatic islets were pulse-labeled for 5 min with [3H]leucine then chased for 25 min, during which time endogenously labeled [3H]proinsulin becomes predominantly compartmented in immature secretory granules. The islets were then homogenized in isotonic sucrose (pH 7.4) and a beta-granule preparation obtained by differential centrifugation and discontinuous sucrose gradient ultracentrifugation. This preparation was enriched 8-fold in beta-granules. Aside from contamination with mitochondria and a limited number of lysosomes, the beta-granule preparation was essentially free of any other organelles involved in proinsulin synthesis and packaging (i.e. microsomal elements and, more particularly, Golgi complex). Conversion of endogenously labeled [3H]proinsulin was followed in this beta-granule fraction for up to 2 h at 37 degrees C in a buffer (pH 7.3) that mimicked the cationic constituents of B-cell cytosol, during which time 92% of the beta-granules remained intact. Proinsulin conversion was analyzed by high performance liquid chromatography. The rate of proinsulin conversion to insulin was stimulated by 2.2 +/- 0.1-fold (n = 6) (at a 60-min incubation) in the presence of ATP (2 mM) and an ATP regenerating system compared to beta-granule preparations incubated without ATP. This ATP stimulation was abolished in the presence of beta-granule proton pump ATPase inhibitors (tributyltin, 2.5 microM, or 1,3-dicyclohexylcarbodiimide, 50 microM). Inhibitors of mitochondrial proton pump ATPases (sodium azide, 20 mM, or oligomycin, 10 micrograms/ml) had no effect on the ATP stimulation of proinsulin conversion. When granules were incubated in a more acidic buffer (pH 5.5), proinsulin conversion was increased relative to that at pH 7.3. At pH 5.5, ATP no longer stimulated conversion, and tributyltin and 1,3-dicyclohexylcarbodiimide had no effect. Disrupted granules only converted proinsulin to a limited extent, and neither ATP nor the inhibitors affected conversion. It is therefore suggested that ATP stimulation of proinsulin conversion in isolated, intact, beta-granules is secondary to intragranular acidification by an ATP-dependent proton pump (reflecting the low pH optimum for proinsulin conversion), rather than ATP dependence of converting activity per se.     

Proinsulin modified by analogues of arginine and lysine is degraded rapidly in pancreatic B-cells.

Modified cytosolic proteins are known to be degraded more rapidly than their native counterparts. In order to determine whether the same applies to a modified protein within the potentially protective environment of secretory granules, rat islets were labelled [( 3H]leucine) in the presence or absence (controls) of 3 mM-canavanine and 3 mM-thialysine (analogues of arginine and lysine respectively), followed by a 24h 'chase' period without analogues. The results showed the following. (1) Incorporation of the analogues into newly synthesized labelled proinsulin inhibited its conversion into insulin during the chase period. (2) Despite this block in conversion, the modified proinsulin was released from islets at the same rate as native proinsulin and insulin from control islets. (3) Morphometric analysis of high-resolution autoradiographs showed that products labelled in the presence of analogues were sequestered into secretory granules at the same rate as native products in control B-cells. (4) Only 7% of prelabelled proinsulin had been degraded within islet cells during the chase period in control islets, compared with 36% for proinsulin prelabelled in the presence of analogues. (5) Control experiments showed that the analogues had no effect on the release or intracellular degradation of unmodified stored insulin (present in islets before exposure to the analogues). (6) Despite sequestration into secretory granules, modified proinsulin, if not released from B-cells, is thus degraded more rapidly than native products.     

Conversion of proinsulin to insulin occurs coordinately with acidification of maturing secretory vesicles

Proinsulin is a single polypeptide chain composed of the B and A subunits of insulin joined by the C-peptide region. Proinsulin is converted to insulin during the maturation of secretory vesicles by the action of two proteases and conversion is inhibited by ionophores that disrupted intracellular H+ gradients. To determine if conversion of prohormone to hormone actually occurs in an acidic secretory vesicle, cultured rat islet cells were incubated in the presence of 3-(2,4- dinitroanilino)-3' amino-N-methyldipropylamine (DAMP), a basic congener of dinitrophenol that concentrates in acidic compartments and is retained there after aldehyde fixation. The cells were processed for indirect protein A-gold colocalization of DAMP, using a monoclonal antibody to dinitrophenol, and proinsulin, using a monoclonal antibody that exclusively reacts with the prohormone. The average density of DAMP-specific gold particles in immature secretory vesicles that contained proinsulin was 71/micron 2 (18 times cytoplasmic background), which indicated that this compartment was acidic. However, the density of DAMP-specific gold particles in the insulin-rich mature secretory vesicle averaged 433/micron 2. This suggests that although proinsulin conversion occurs in an acidic compartment, the secretory vesicles become more acidic as they mature. Since the concentration of anti- proinsulin IgG binding in secretory vesicles is inversely proportional to the conversion of proinsulin to insulin, we were able to determine that maturing secretory vesicles had to reach a critical pH before proinsulin conversion occurred.     

Proinsulin precursors in catfish pancreatic islets.

The purpose of these experiments was to determine whether insulin-related peptides, larger than proinsulin, could be detected in pancreatic islet cells. Catfish pancreatic islets were incubated with radiolabeled amino acids. After 15- to 60-min incubation, two acid-alcohol-extractable peptides, larger than proinsulin, were detected which were approximately of Mr = 12,000 and 11,000 (12 K and 11K, respectively). They migrated as single polypeptide chains by sodium dodecyl sulfate-urea polyacrylamide gel electrophoresis under reducing conditions, and were therefore not aggregates of insulin or proinsulin. The 12 K protein had identical mobility with catfish preoproinsulin synthesized in a wheat germ cell-free system. On standard electrophoresis at pH 8.9, the 12 K protein migrated separately from proinsulin and was at least 65% one protein with two to three minor contaminants. The 12 K and 11 K proteins were chemically related to insulin and proinsulin as shown by tryptic peptide analysis, using cation exchange resin chromatography, and by two-dimensional tryptic peptide maps. Analysis of the tryptic digest of the 12 K protein, compared to proinsulin after leucine aminopeptidase treatment, suggested that the NH2 terminus of the larger protein was different from that of proinsulin. These peptides were specifically bound to anti-insulin antibody. The binding was only 5 to 8% of the protein added, but was specific for the 12 K and 11 K proteins when the immunoprecipitates were examined by electrophoresis and not from contaminating proinsulin. During the continuous incubation of the islets with [3H]leucine, 12 K and 11 K proteins were synthesized in the cell before proinsulin. When islets were first incubated with [3H]leucine for 30 min followed by incubation with excess unlabeled leucine, the 12 K and 11 K proteins appeared to show a precursor-product relationship to proinsulin and insulin. Even when total islet protein synthesis was inhibited by cycloheximide (100 microgram/ml), proinsulin continued to be synthesized for up to 2 h. This suggested that the conversion of the proinsulin precursors to proinsulin in the fish is a post-translational event.     

Glucose stimulates the biosynthesis of rat I and II insulin to an equal extent in isolated pancreatic islets

The effects of glucose on insulin biosynthesis were studied by measuring the incorporation of radiolabelled amino acids into proinsulin/insulin in isolated rat islets. The islets were pulse labelled for 15 min with [3H]leucine (present in rat insulin I and II) or [35S]methionine (unique to rat insulin II) and then incubated for a 165 min post-label (chase) period during which the majority of labelled proinsulin was converted to insulin but under conditions whereby greater than 95% of radiolabelled proinsulin or insulin was retained in the islets. The newly synthesized, labelled, insulin was analyzed by high performance liquid chromatography. Rat I and II insulin biosynthesis was stimulated by 16.7 mM glucose to the same extent.     

Metabolic signals produced by purine ribonucleosides stimulate proinsulin biosynthesis and insulin secretion.

Inosine, guanosine and adenosine strongly stimulated proinsulin biosynthesis and insulin secretion in isolated mouse pancreatic islets. None of the purine ribonucleosides stimulated insulin secretion in rat islets, although as reported [jain & Logothetopoulos (1977) Endocrinilogy 100, 923-927] inosine and guanosine, but no adenosine, were potent stimulants of proinsulin biosynthesis in this species. The purine bases had no effect in either species. D-Ribose, which enhanced proinsulin biosynthesis at 0.3 and 0.6 mM but not at 5mM in rat pancreatic islets [jain & Logothetopoulos (1977) Endocrinology 100, 923-927], produced no secretory signals in rat islets and was without any effect on proinsulin biosynthesis and insulin secretion in mouse islets. The rates of oxidation of 14C-labelled purine ribonucleosides and D-ribose in islets of the two species correlated well with their effectiveness as inducers of insulin secretion and proinsulin biosynthesis. Specific inhibitors of purine ribonucleoside phosphorylase, adenosine deaminiase and of purine ribonucleoside transport suppressed the stimulatory effects of nucleosides in pancreatic islets without altering the effect of D-glucose. The same inhibitors also markedly diminished the oxidation rats of the labelled purine ribonucleosides. The experiments clearly indicate that porinsulin biosynthesis and insulin secretion are modulated through metabolic signals and not through interactions of intact substrate molecules with cell receptors.     

Identification of the structures of multiple subspecies of protein kinase C expressed in rat brain

Synthesis and processing of radiolabelled rat insulin I and II were studied by pulse-labelling freshly isolated rat islets with [3H]leucine and chasing in 2 mM glucose for up to 270 min (which minimized insulin secretion, less than 1%/h). Islet samples were taken during the chase period and analyzed for their rat insulin I and II content by high-performance liquid chromatography. Prior to 60 min chase rat insulin I accounted for greater than 85% of the radiolabelled insulin present. With longer periods of chase, the relative percentage of rat insulin II progressively increased so that by completion of proinsulin to insulin processing the two labelled rat insulins were present in the same proportion as the relative immunoreactive content, approx. 60:40% insulin I/insulin II. Thus, although islets synthesize the two insulins in proportion to their relative immunoreactive content, rat insulin I and II are processed with different kinetics.     

Preferential cleavage of des-31,32-proinsulin over intact proinsulin by the insulin secretory granule type II endopeptidase. Implication of a favored route for prohormone processing.

Two Ca(2+)-dependent endopeptidase activities are involved in proinsulin to insulin conversion: type I cleaves COOH-terminal to proinsulin Arg31-Arg32 (B-chain/C-peptide junction); and type II preferentially cleaves at the Lys64-Arg65 site (C-peptide/A-chain junction). To further understand the mechanism of proinsulin processing, we have investigated types I and II endopeptidase processing of intact proinsulin in parallel to that of the conversion intermediates, des-31,32-proinsulin and des-64,65-proinsulin. The type I processed des-64,65-proinsulin and proinsulin at the same rate. In contrast, the type II endopeptidase processed des-31,32-proinsulin at a much faster rate (> 19-fold; p < 0.001) than it did intact proinsulin. Furthermore, unlabeled proinsulin concentrations required for competitive inhibition of 125I-labeled des-64,65-proinsulin and 125I-proinsulin processing by a purified insulin secretory granule lysate were similar (ID50 = 14-16 microM), whereas inhibition of 125I-labeled des-31,32-proinsulin processing required a higher nonradiolabeled proinsulin concentration (ID50 = 197 microM). Synthetic peptides corresponding to the sequences surrounding Lys64-Arg65 (AC-peptide/substrate) and Arg31-Arg32 (BC-peptide/substrate) of human proinsulin were synthesized for use as specific substrates or competitive inhibitors. Cleavage of the BC-substrate by type I and AC-substrate by type II was COOH-terminal of the dibasic sequence, with similar Ca(2+)-and pH requirements previously observed for proinsulin cleavage. Apparent Km and Vmax for type I processing of the BC-substrate was Km = 20 microM; Vmax = 22.8 pmol/min, and for type II processing of the AC-substrate was Km = 68 microM; Vmax = 97 pmol/min. In competitive inhibition assays, the BC-peptide similarly blocked insulin secretory granule lysate processing of des-64,65-proinsulin and proinsulin (ID50 = 45-55 microM), but did not inhibit des-31,32-proinsulin processing. However, the AC-peptide preferentially inhibited insulin secretory granule lysate processing of des-31,32-proinsulin (ID50 = microM) compared to proinsulin (ID50 = 330 microM), and not des-64,65-proinsulin. We conclude that the type I endopeptidase recognized des-64,65-proinsulin and proinsulin as similar substrates, whereas the type II endopeptidase has a stronger preference for des-31,32-proinsulin compared to intact proinsulin. Furthermore, we suggest that in intact proinsulin there exists a constraint to efficient processing that is relieved following type I processing. Structural flexibility, in addition to the presence of Lys64-Arg65, therefore appears to be important for type II endopeptidase specificity and may provide a molecular basis for a preferential route of proinsulin conversion via des-31,32-proinsulin.     

Viability tests of cryopreserved endocrine pancreatic cells

Collagenase-isolated islets, which had been cultured for 1 week, were frozen at two different cooling rates. Islets frozen at 5 degrees C/min behaved from a functional point of view very similarly to that of nonfrozen, cultured control islets, except for a reduced maximal insulin secretory capacity and a reduced insulin content. Slowly frozen islets (0.5 degrees C/min), however, displayed reduced rates of both proinsulin biosynthesis and glucose oxidation. It is concluded that isolated islets can be cryopreserved with great success and that the methods of choice for viability tests are those characterizing the dynamics of insulin secretory capacity of the cryopreserved islets.     

Model of synthesis and release of insulin from rat islet β-cells and the effect of pretreatment with tolbutamide

A kinetic model involving synthesis of proinsulin in the rough endoplasmic reticulum, maturation through the Golgi apparatus and granules, with conversion to insulin, is proposed to account for data on the amount of insulin and of proinsulin both secreted during various time intervals and remaining in islets. Introducing three compartments for granules makes it possible to account for the measurement of both hot (pulse labeled with tritiated leucine) and cold proinsulin and insulin over a period of 21/2 hr under constant glucose. Data from islets from animals pretreated with tolbutamide are also presented and modeled. The model is then expanded so that it can be successfully applied to available data on the effects of a period of glucose deprivation on secretion of both hot and cold hormone. Parameters have essentially the same values, where they overlap, as were obtained (Landahl and Grodsky, 1982Bull. math. Biol. 44, 399–410) from insulin secretion by perfused rat pancreas stimulated by a variety of temporal patterns of glucose concentration.     

Insulin receptors on rat pancreatic islets: specificity of 125 I-insulin binding

Binding sites of isolated rat pancreatic islets have been shown to interact with insulin. Employing various species-insulins, insulin analogues and substances not being structurally related to insulin, structure-specificity as well as pH- and temperature-dependence of insulin binding to rat pancreatic islets have been studied. Rat insulin displaced 125 I-insulin from its binding sites in the same concentration-dependent manner as pork insulin did, whereas the insulin analogue des-(phe-val-asp)B1-3-p-glu B4-insulin was less effective. Pork C-peptide hardly competed for binding and pork proinsulin did not compete at all. Both the species' insulins inhibited glucose (16.7 mM)-induced insulin secretion. The inhibitory effect was less when des-(phe-val-asp)B1-3-p-glu B4-insulin was employed and no inhibition of insulin secretion was observed by the use of pork C-peptide or proinsulin. Glucagon and somatostatin did not affect insulin binding. pH optimum of insulin binding appears to be in the range between 7.0 and 8.0. Binding was augmented with increasing temperature up to 37 degrees C. It is concluded that rat pancreatic islets possess insulin because binding and biological potency of substances related to insulin were in harmony. Moreover pH- and temperature-optimum of insulin binding are in a physiological range.     

Sulfatide promotes the folding of proinsulin, preserves insulin crystals, and mediates its monomerization.

Sulfatide is a glycolipid that has been associated with insulin-dependent diabetes mellitus. It is present in the islets of Langerhans and follows the same intracellular route as insulin. However, the role of sulfatide in the beta cell has been unclear. Here we present evidence suggesting that sulfatide promotes the folding of reduced proinsulin, indicating that sulfatide possesses molecular chaperone activity. Sulfatide associates with insulin by binding to the insulin domain A8--A10 and most likely by interacting with the hydrophobic side chains of the dimer-forming part of the insulin B-chain. Sulfatide has a dual effect on insulin. It substantially reduces deterioration of insulin hexamer crystals at pH 5.5, conferring stability comparable to those in beta cell granules. Sulfatide also mediates the conversion of insulin hexamers to the biological active monomers at neutral pH, the pH at the beta-cell surface. Finally, we report that inhibition of sulfatide synthesis with chloroquine and fumonisine B1 leads to inhibition of insulin granule formation in vivo. Our observations suggest that sulfatide plays a key role in the folding of proinsulin, in the maintenance of insulin structure, and in the monomerization process.     

Proinsulin biosynthesis in broken-cell preparations of islets of Langerhans.

1. Rabbit islets of Langerhans were disrupted by ultrasonic methods and the sonicated preparations were used to study proinsulin biosynthesis. 2. When [3h]leucine is incubated in such preparations, incorporation takes place into proinsulin, as evidenced by characterization on polyacrylamide gels, and by the conversion of this labelled material into insulin, by using trypsin. 3. The labelled proinsulin may also be purified by antiinsulin antibody bound to Sepharose. 4. With the broken-cell preparation it was shown that incorporation of leucine is accelerated by increasing the glucose content of the medium from 2mM to 16mM. However, 16mM-galactose or -sucrose did not stimulate incorporation significantly from basal values. This effect of glucose was abolished by cycloheximide. 5. The significance of these findings in relation to the mechanism of glucose stimulation of proinsulin biosynthesis is discussed.     

Stimulation of proinsulin biosynthesis and insulin release by pyruvate and lactate.

Increasing concentrations of pyruvate failed to stimulate proinsulin biosynthesis and insulin release in freshly isolated islets. Glycolytic flux (3H2O from [5-3H]glucose) decreased by 80-85%, but decarboxylation of [1(-14)C]pyruvate was unaffected in islets tested immediately after alloxan exposure. This strongly suggested that in freshly isolated islets, beta-cells, in relation to other islet cells, hardly contribute to the decarboxylation of pyruvate. Non-alloxan-treated cultured islets decarboxylated 2-2.5 times as much pyruvate as did alloxan-treated islets cultured for 15-18h. Thus the contribution of beta-cells to the metabolism of pyruvate after culturing markedly increased. Concomitantly beta-cells became responsive to pyruvate. At 20mM-pyruvate, release of prelabelled proinsulin and insulin and incorporation of [3H]leucine into proinsulin reached values approximately half of those obtained with 20mM-glucose. Lactate was as effective as pyruvate in inducing responses in cultured islets. The experiments indicate that a critical degree of substrate utilization is necessary for the generation of signals for insulin release and proinsulin biosynthesis.     

Transglutaminase and cellular motile events: Retardation of proinsulin conversion by glycine methylester

Glycine methylester, an inhibitor of transglutaminase, decreased glucose-stimulated insulin release and delayed proinsulin conversion in rat pancreatic islets pulselabelle with L-[4-[³H]phenylalanine. Sarcosine methylester, which does not inhibit transglutaminase activity, failed to affect insulin release and proinsulin conversion. The incorporation of L-[4-³H]phenylalanine into islet peptides, the ratio of hormonal to total tritiated peptides and the insulin content of the islets failed to be affected by either of these methylesters. It is proposed that transglutaminase participates in the control of motile events involved in both the transfer of proinsulin from its site of synthesis to its site of conversion, and the translocation of insulin from its site of storage to its site of release.     

The intracellular handling of insulin-related peptides in isolated pancreatic islets. Evidence for differential rates of degradation of insulin and C-peptide.

Islets of Langerhans isolated from adult rats were maintained in tissue culture for 3 days in the continued presence of [3H]leucine. Labelled proinsulin, C-peptide and insulin were measured by quantitative h.p.l.c., a method which also allowed for resolution of C-peptide I and II, and of insulin I and II (the products of the two rat insulin genes). The results showed that: (1) at early times, proinsulin was the major radiolabelled product; with progressive time in culture, intra-islet levels of [3H]proinsulin decreased, despite continuous labelling with [3H]leucine, indicating that the combined rates of proinsulin conversion into insulin and of proinsulin release, exceeded the rate of synthesis; (2) insulin I levels were always greater than those of insulin II, both in the islets and for products released to the medium; (3) the molar ratio of [3H]insulin I and II to their respective 3H-labelled C-peptides increased with time for products retained within islets, reaching a value close to 3:1 by 3 days; by contrast, for products released to the medium during the culture period, the ratio was always close to unity; (4) when islets were incubated with [3H]leucine for 2 days, and then left for a further 1 day without label (chase period), the intra-islet [3H]insulin/[3H]C-peptide ratios rose to values as high as 9:1. Again, for material released to the medium, the values were close to 1:1; (5) it is concluded that C-peptide is degraded more rapidly than insulin within islet cells, thereby accounting for the elevated insulin/C-peptide ratios. The difference between the ratios observed in the islets and those for material released to the medium is taken to indicate that degradation occurs in a discrete cellular compartment and not in the secretory granule itself.     

Proinsulin disulfide maturation and misfolding in the endoplasmic reticulum

Upon nonreducing Tris-Tricine-urea-SDS-PAGE, newly synthesized proinsulin from pancreatic islets of normal rodents forms a band fast mobility representing the native disulfide isomer, which is efficiently secreted. In addition at least two slower migrating "isomer 1 and 2" bands are recovered, not discernible under reducing conditions, which represent minor species that exhibit less efficient secretion. Although rats and mice have two proinsulin genes, three distinct migrating species are also produced upon proinsulin expression from a single wild-type human proinsulin cDNA. The "Akita-type" proinsulin mutation, which causes dominant-negative diabetes mellitus due to point mutation C(A7)Y that leaves B7-cysteine without its disulfide pairing partner, is recovered as a form that near quantitatively co-migrates with the aberrant isomer 1 band of proinsulin. Anomalous migration is also demonstrated for several other mutants lacking a single cysteine. In islets from PERK-/- mice, which exhibit premature loss of pancreatic beta cells, hypersynthesis of proinsulin increases the amount of nonnative proinsulin isomers. Such findings appear consistent with an hypothesis that supranormal production of nonnative proinsulin may predispose to pancreatic beta cell toxicity.