Hyperproinsulinaemia in obese fat/fat mice associated with a carboxypeptidase E mutation which reduces enzyme activity.

Mice homozygous for the fat mutation develop obesity and hyperglycaemia that can be suppressed by treatment with exogenous insulin. The fat mutation maps to mouse chromosome 8, very close to the gene for carboxypeptidase E (Cpe), which encodes an enzyme (CPE) that processes prohormone intermediates such as proinsulin. We now demonstrate a defect in proinsulin processing associated with the virtual absence of CPE activity in extracts of fat/fat pancreatic islets and pituitaries. A single Ser202Pro mutation distinguishes the mutant Cpe allele, and abolishes enzymatic activity in vitro. Thus, the fat mutation represents the first demonstration of an obesity-diabetes syndrome elicited by a genetic defect in a prohormone processing pathway.

Preparation and characterization of plasma membrane-enriched fractions from rat pancreatic islets.

Methods have been developed for the isolation on a semi-micro scale of a plasma membrane-enriched fraction from rat islets of Langerhans. An important feature of these experiments is the use of 125I-labeled wheat germ agglutinin as a specific probe for plasma membrane-containing fractions. The partly purified plasma membrane fraction had a density in sucrose of about 1.10 and was enriched in the activities of 5'-nucleotidase, alkaline phosphatase, sodium-potassium, and magnesium-dependent ATPase and adenylate cyclase. It contained only very low levels of acid phosphatase, cytochrome c oxidase, insulin, and RNA. Further purification was hampered by the relatively small amounts of fresh plasma membrane material that could be obtained from 16-24 rats in each experiment. When islets were prelabeled with radioactive fucose, the plasma membrane-enriched fraction contained radioactivity at a four- to fivefold higher specific acivity than the whole islet homogenate. Sodium dodecyl sulfate (SDS) polyacrylamide gel electrophoresis of plasma membrane-enriched fractions pooled from several experiments revealed a distinctive pattern of protein bands as compared with other less pure fractions. With respect to rapidity, apparent specificity, and easy reversibility of the labeling of the plasma membrane fraction, 125I-wheat germ agglutinin provides a highly useful tool for the detection of microgram quantities of plasma membrane components which should be applicable to many other systems as well.

Folding of insulin receptor monomers is facilitated by the molecular chaperones calnexin and calreticulin and impaired by rapid dimerization.

Many complex membrane proteins undergo subunit folding and assembly in the ER before transport to the cell surface. Receptors for insulin and insulin-like growth factor I, both integral membrane proteins and members of the family of receptor tyrosine kinases (RTKs), are unusual in that they require homodimerization before export from the ER. To better understand chaperone mechanisms in endogenous membrane protein assembly in living cells, we have examined the folding, assembly, and transport of the human insulin receptor (HIR), a dimeric RTK. Using pulse-chase labeling and nonreducing SDS-PAGE analysis, we have explored the molecular basis of several sequential maturation steps during receptor biosynthesis. Under normal growth conditions, newly synthesized receptor monomers undergo disulfide bond formation while associated with the homologous chaperones calnexin (Cnx) and calreticulin (Crt). An inhibitor of glucose trimming, castanospermine (CST), abolished binding to Cnx/Crt but also unexpectedly accelerated receptor homodimerization resulting in misfolded oligomeric proreceptors whose processing was delayed and cell surface expression was also decreased by approximately 30%. Prematurely-dimerized receptors were retained in the ER and more avidly associated with the heat shock protein of 70 kD homologue binding protein. In CST-treated cells, receptor misfolding followed disordered oligomerization. Together, these studies demonstrate a chaperone function for Cnx/Crt in HIR folding in vivo and also provide evidence that folding efficiency and homodimerization are counterbalanced.

Cloned cell lines from a transplantable islet cell tumor are heterogeneous and express cholecystokinin in addition to islet hormones.

A liver metastasis (MSL) with a remarkable in vitro proliferation potential has been identified in an NEDH rat carrying a transplantable x-ray-induced islet cell tumor. Two insulin-secreting cell lines, MSL-G and MSL-H, with doubling times of 3-5 d were established by repeated limiting dilution cloning. In vivo inoculation of MSL-G cells induced severe hypoglycemia caused by a small but highly heterogeneous tumor as revealed by immunocytochemistry. Whereas most cells stained for the islet hormones, insulin, glucagon, and somatostatin, clustered cells were discovered to contain cholecystokinin (CCK). Additional in vitro-limiting dilution cloning, followed by immunocytochemical characterization, clearly demonstrated the capacity of single cell clones to simultaneously express the same four hormones. Radioimmunoassays with a panel of site-specific antisera of culture supernatants and purified cell extracts showed the MSL-G2 cells to produce, store, and secrete readily detectable amounts of processed and unprocessed CCK. Gastrin was not detected while coexpression of glucagon and CCK were demonstrated. Mutant clones selected for resistance to 6-thioguanine (frequency, 2 X 10(-7] and checked for HAT (hypoxanthine, aminopterin, thymidine) sensitivity retained the capacity for multi-hormone expression. We propose that the MSL tumor contains pluripotent endocrine stem cells. The MSL tumor and the MSL-G2 cells in particular will allow studies of not only CCK biosynthesis and processing but also of mechanisms involved in tumor and islet cell differentiation.

Insulin biosynthesis: evidence for a precursor.

Human islet cell tumor tissue and isolated islets of Langerhans from rats incorporated radioactive amino acids in vitro into insulin and a larger acid-alcohol soluble protein which could be separated from insulin by gel filtration. The amino acids were incorporated into the larger protein earlier than into insulin; only after incubation of islets for approximately 30 minutes did radioactivity begin to appear in insulin. The transfer of about 70 percent of the radioactivity of the larger protein to insulin was demonstrated in the absence of new peptide bond synthesis (cycloheximide), or during incubation with unlabeled amino acid (chase). The results indicate that the larger protein is a precursor in the biosynthesis of insulin. The name "proinsulin" is suggested for this protein.

Insulin-resistant diabetes due to a point mutation that prevents insulin proreceptor processing.

A point mutation in the human insulin receptor gene in a patient with type A insulin resistance alters the amino acid sequence within the tetrabasic processing site of the proreceptor molecule from Arg-Lys-Arg-Arg to Arg-Lys-Arg-Ser. Epstein-Barr virus-transformed lymphocytes from this patient synthesize an insulin receptor precursor that is normally glycosylated and inserted into the plasma membrane but is not cleaved to mature alpha and beta subunits. Insulin binding to these cells is severely reduced but can be increased about fivefold by gentle treatment with trypsin, accompanied by the appearance of normal alpha subunits. These results indicate that proteolysis of the proreceptor is necessary for its normal full insulin-binding sensitivity and signal-transducing activity and that a cellular protease that is more stringent in its specificity than trypsin is required to process the receptor precursor.

A brief perspective on insulin production.

The preeminent role of the beta cell is to manufacture, store and release insulin. The mature insulin molecule is composed of two polypeptide chains designated as A and B that are joined by two pairs of disulfide bonds with an additional intramolecular disulfide bond in the A chain. However, the two chains of the insulin molecule are not synthesized as separate polypeptide chains but rather are generated by specific proteolytic processing of a larger precursor, proinsulin. This discovery in 1967 and the concept of prohormones changed our view of the biosynthesis of hormones and neuropeptides. It allowed studies of the regulation of insulin biosynthesis that highlighted the key role of glucose. In addition, the C-peptide, the polypeptide that joins the A and B chains in proinsulin and is stored with insulin in the secretory granules and secreted in equimolar amounts, allowed studies of pancreatic beta cell function in vivo including in patients with diabetes. Subsequent studies have identified the specific proteases, prohormone convertases 1/3 and 2 and carboxypeptidase E, that are involved in the conversion of proinsulin to proinsulin intermediates and then to insulin. Disorders of (pro)insulin biosynthesis continue to illuminate important aspects of this pathway, revealing important connections to diabetes pathogenesis. Recent studies of patients with insulin gene mutations that cause permanent neonatal diabetes have identified key residues affecting the folding and structural organization of the preproinsulin molecule and its subsequent processing. These findings have renewed interest in the key role of endoplasmic reticulum function in insulin biosynthesis and the maintainance of normal beta cell health.

Furin mediates enhanced production of fibrillogenic ABri peptides in familial British dementia.

The genetic lesion underlying familial British dementia (FBD), an autosomal dominant neurodegenerative disorder, is a T-A transversion at the termination codon of the BRI gene. The mutant gene encodes BRI-L, the precursor of ABri peptides that accumulate in amyloid deposits in FBD brain. We now report that both BRI-L and its wild-type counterpart, BRI, were constitutively processed by the proprotein convertase, furin, resulting in the secretion of carboxyl-terminal peptides that encompass all or part of ABri. Elevated levels of peptides were generated from the mutant BRI precursor. Electron microscopic studies revealed that synthetic ABri peptides assembled into irregular, short fibrils. Collectively, our results support the view that enhanced furin-mediated processing of mutant BRI generates fibrillogenic peptides that initiate the pathogenesis of FBD.

Studies on the secretion of newly synthesized proinsulin and insulin from isolated rat islets of Langerhans.

Islets of Langerhans isolated from rat pancreas were incubated at 37 degrees C(95% O(2)/5% CO(2)) in buffered medium containing 1.0 mg/ml glucose and leucine (3)H for 1 hr (1st hr), washed, and incubated for an additional hr (2nd hr) in low glucose medium (0.5-1.0 mg/ml) containing unlabeled leucine. A portion of the islets was then extracted with acid-ethanol and the remainder were transferred to medium containing 3.0 mg/ml glucose and incubated for 2 hr (3rd and 4th hr) at 37 degrees C. The medium was exchanged at 30-min intervals and portions of the islets were extracted at the 3rd and 4th hr. The total amounts and specific activities of the proinsulin and insulin in the islet extracts and medium samples were determined after fractionation on Biogel P-30 columns in 3 M acetic acid. Maximal release of newly synthesized insulin occurred between the 3rd and 4th hr of incubation, confirming the results of Howell and Taylor (Biochem. J.102: 922. 1967). The high glucose medium increased the secretion of insulin approximately three to fourfold. The ratio of the specific activities of the insulin in the medium to that in the islets was about 1/1 during incubation in low glucose, but it increased to 2.5/1 during incubation with high glucose. The peak occurred at the 3rd hr, i.e., 1 hr after exposure to high glucose. The ratio of labeled proinsulin to insulin was slightly lower in the medium than in the islets. Addition of sufficient cycloheximide after the 1st hr to inhibit protein synthesis did not inhibit these responses. The specific activity of the proinsulin in the medium was about the same as that in the islets, and both were about 10-fold higher than the specific activity of the insulin. High glucose did not alter the proinsulin specific activity, which tended to decline throughout the period of observation. With cycloheximide present, the concentration of proinsulin in the islets steadily declined while the specific activity of proinsulin remained high, indicating that the proinsulin pool is small and is turning over rapidly. In terms both of amount and radioactivity proinsulin amounted to 6-7% on a molar basis of the insulin in both the medium and the islets. Addition of dibutyryl cyclic 3',5'-adenosine monophosphate (DBCAMP) (0.002 M) with high glucose during the postlabeling period slightly increased the rate of insulin secretion (133% of control) but did not significantly alter the other parameters. The results suggest that while newly synthesized insulin and proinsulin may be preferentially secreted to a slight degree, about 90% of the insulin released during 3 hr in response to glucose, or to glucose and DBCAMP, is derived from pre-existing granule stores. There were no indications of the existence of independent or nongranule pathways of insulin or proinsulin secretion.

Retention and degradation of 125I-insulin by perfused livers from diabetic rats.

The retention of degradation of insulin by isolated perfused liver have been examined. Noncyclically perfused livers from streptozotocin-diabetic rats retained 25% and degraded 10% of 125I-insulin administered as a 1-min pulse. On gel filtration (Sephadex G50F), the degradation products released into the vascular effluent eluted in the salt peak. During the 45-min interval after the end of the 125I-insulin infusion, 0.19% of the total dose was excreted in the bile. 60-90% of this material consisted of iodinated, low-molecular-weight degradation products. Inclusion of native insulin with the 125I-insulin in the pulse depressed both the retention and degradation of iodinated material; however, this reflected increased retention and degradation of the total insulin dose (125I-insulin plus native hormone). The log of the total amounts of insulin retained and degraded were linearly related to the log of the total amount of insulin infused at concentrations between 12.7 nM and 2.84 muM. Increasing the amount of native insulin in the infused pulse also depressed the total amount of iodinated material found in the bile and led to the appearance in the bile of intermediate-sized degradation products that did not simultaneously appear in the vascular effluent. Addition of high concentrations of glucagon to the infused 125I-insulin had no effect on the retention or degradation of the labeled hormone, or on the apparent size and amount of iodinated degradation products found in the vascular effluent or in the bile. Preinfusion of concanavalin A inhibited both 125I-insulin retention and degradation. A greater depression by concanavalin A of degradation than binding was also observed with isolated hepatocytes. In contrast to 125I-insulin, the retention and degradation of two iodinated insulin analogues of relative low biological potency, proinsulin and desalanyl-desasparaginyl insulin, were small. The amount of radioactivity appearing in the bile after infusion of these analogues was almost negligible. However, degradation products of these analogues that appeared in the bile and in the vascular effluent was qualitatively similar to those found after the infusion of 125I-insulin. Our findings suggest that the rapid initial uptake of 125I-insulin after its infusion into noncyclically perfused liver, as well as its subsequent degradation, behaves in a qualitatively similar fashion to the binding of 125I-insulin and its degradation by isolated rat hepatocytes. This uptake and the subsequent phase of degradation may be attributable to binding of insulin at specific recognition sites, preliminary to its transfer to a degradative site(s) presumed to be located inside the cell.

Human serum proinsulin.

Gel filtration of human serum extracts on Bio-Gel P-30 columns produced two peaks of material reactive with insulin antisera. The earlier eluting fraction appeared at the elution position of proinsulin (serum proinsulin-like component, PLC) while the second fraction corresponded in elution volume to insulin. In assays using porcine insulin-(131)I and an antiserum against porcine insulin, human pancreatic proinsulin was less reactive than human insulin. Serial dilutions of the serum PLC in the immunoassay showed immunological identity with the human proinsulin standard. Partial tryptic digestion of the serum PLC yielded products with increased immunological reactivity as estimated with insulin as the standard. With larger amounts of trypsin, all the serum PLC was converted to insulin-like components (desthreonine and desoctapeptide insulin). On the basis of these results we conclude that the earlier eluting fraction of human serum extracts is proinsulin. The fasting values of proinsulin in normal subjects ranged between 0.05 and 0.4 ng/ml, representing from 5 to 48% of the insulin concentration. In one subject the values of proinsulin were higher than those of insulin. After oral administration of 100 g of glucose, the proinsulin levels tended to rise similarly to insulin. Three obese patients with hyperinsulinemia had higher fasting levels of proinsulin and a greater increase after glucose than the normal subjects. As the high levels of proinsulin coexisted with raised insulin concentration in these obese subjects, the relative proportions of the two hormones were in the same range observed in the normal group. Thus hyperinsulinemia in these obese subjects was not accompanied by an increase in the fraction of serum proinsulin. When the values for serum proinsulin were expressed as percentage of the insulin levels, there was a decrease in the per cent proinsulin in the first hour of the glucose tolerance test. After the second hour, the per cent tended to rise towards the fasting levels.

The metabolism of proinsulin and insulin by the liver.

The removal of bovine proinsulin by the isolated perfused rat liver has been studied and the results compared with the removal of insulin. At high concentrations of insulin (> 180 ng/ml) the removal process was saturated and the t(1/2) varied between 35 and 56 min. With low initial insulin levels the disappearance followed first-order kinetics, the mean regression coefficient being - 0.022, t(1/2) 13.8 min, and the hepatic extraction 4.0 ml/min. The results with proinsulin were in striking contrast to these findings. At both high and low concentrations the hepatic removal of proinsulin was considerably slower, averaging 10-15 times less than that of insulin. Specific immunoassay techniques and gel filtration of samples taken from perfusions to which both labeled and unlabeled proinsulin had been added did not show conversion to either insulin or the C-peptide. Bovine and rat (131)I-labeled proinsulins were degraded more slowly than bovine insulin-(131)I by bovine and rat liver homogenates. Both proinsulin and insulin inhibited the degradation of insulin-(131)I, equimolar quantities of proinsulin being 2-5 times less effective than insulin. These results indicate significant differences in the capacity of the liver to remove and degrade insulin and proinsulin. The low hepatic extraction of proinsulin may account for its prolonged half-life in vivo and contribute to its relatively high plasma concentration in the fasting state. Furthermore this finding will have to be taken into account in the interpretation of changes in the proinsulin:insulin ratios in peripheral blood in a variety of metabolich situations.

Expression of five cathepsins in murine melanomas of varying metastatic potential and normal tissues.

The relative levels of mRNAs for cathepsins B, D, H, L, and S in eight normal murine tissues and three murine melanoma variants, B16-F1, B16-F10, and B16a, have been analyzed by RNA dot blot and densitometry. A direct correlation was observed between the levels of cathepsin B mRNA and the metastatic potentials of these three melanoma variants. The relative amount of cathepsin B mRNA in B16a, which is the melanoma variant with the highest metastatic potential, was at least 3 times greater than that found in any of the normal murine tissues surveyed. Similar results were obtained in analyses of either solid tumors or of cultures of tumor cells, confirming that the tumor cells themselves were the source for the elevated expression of cathepsin B mRNA. Northern blot analysis revealed the presence of three cathepsin B transcripts of 5.0, 4.0, and 2.2 kilobases in the melanoma variants, while only the 2.2-kilobase transcript was seen in the normal murine tissues. Concurrently with the mRNA analysis, enzyme assays for cathepsin B activity were also performed using synthetic peptide substrates. The assays revealed increased cathepsin B activities in the melanoma variants, corresponding well with the increased cathepsin B mRNA levels, and in addition demonstrated that all three of the melanoma variants secreted a latent form of cathepsin B into conditioned medium, which could be activated by limited proteolysis with pepsin. The levels of the latent enzyme released by the murine melanoma variants correlated well with the levels of cathepsin B mRNA and with the metastatic potentials as determined by spontaneous metastasis form a s.c. site.

Evolution of the prohormone convertases: identification of a homologue of PC6 in the protochordate amphioxus.

Many of the protein precursors traversing the secretory pathway undergo cleavage at multibasic sites to generate their bioactive forms. The proprotein convertases (PCs), a family of subtilisin-like proteases, are the major endoproteases that serve this function. Genes encoding seven distinct members of this family have so far been characterized in vertebrates: furin, PC2, PC1/PC3, PC4, PACE4, PC5/PC6 and PC7/PC8/LPC. Multiple PC genes have also been cloned from a number of invertebrates, including Drosophila melanogaster and Caenorhabditis elegans. These findings suggest that gene duplication and diversification of the PCs have occurred throughout metazoan evolution. To investigate the structural and functional changes which have occurred during vertebrate development, we have analyzed the expression of PC genes in the protochordate amphioxus. We have previously shown that amphioxus express homologous PC2 and PC1/PC3 genes [Proc. Natl. Acad. Sci. USA 92 (1995) 3591]. Here we report the characterization of amphioxus cDNAs encoding proteases with a high degree of similarity to mammalian PC6. Three cDNAs encoding three PC6 isoforms differing only in their carboxy-terminal sequences were found, derived by alternative splicing. Two isoforms appear to be soluble enzymes, whereas the third contains a transmembrane hydrophobic segment and thus is likely to be membrane-bound. All three variants contain many repeats of a cysteine-rich motif that is found in several other PC family members. Thus, amphioxus, like the vertebrates, expresses two types of PCs, e.g., PC2 and PC1/PC3 which function in the regulated secretory pathway in neuroendocrine cells, and the more widely expressed PC6 which functions mainly in the constitutive pathway.

Characterization of multiple prohormone convertase PC1/3 transcripts in porcine ovary.

Overlapping cDNAs encoding porcine prohormone convertase, PC1/3, have been isolated from a pregnant sow ovary cDNA library using a mouse PC1/3 cDNA as a probe. Nucleotide sequence analysis of these cDNAs predicts a PC1/3 precursor protein of 753 amino acid residues, which shares an overall sequence homology of 96, 92, and 92% with the human, rat, and mouse counterparts, respectively. Furthermore, five different polyadenylation sites have been observed. The utilization of these polyadenylation sites results in a length difference of 40-440 bp in the 3' untranslated regions of the transcripts.

On the role of the proinsulin C-peptide.

Intracellular cleavage of protein and polypeptide precursors is now recognized as a widely occurring biosynthetic mechanism. As this field has developed, proinsulin and its cleavage patterns and secretory products have served as useful models for investigations of other systems. A particularly relevant aspect of the proprotein concept is the simple mechanism it provides for the coördinate synthesis and discharge of related peptides from endocrine or other secretory cells. This report reviews briefly the role of the proinsulin C-peptide, first in terms of its special biosynthetic functions, which are unique to the assembly of the two-chain insulin structure, and then with regard to its more general implications for other biosynthetic and secretory systems.

Is islet amyloid polypeptide a significant factor in pathogenesis or pathophysiology of diabetes?

Islet amyloid polypeptide (IAPP) or amylin, a recently discovered minor secretory peptide of the beta-cell related to calcitonin gene-related peptide (CGRP), is a constituent of amyloid deposits in the islets of many non-insulin-dependent (type II) diabetic individuals and some elderly nondiabetic subjects. IAPP is synthesized as a small precursor at a level of approximately 1% that of insulin and is processed, amidated, stored in beta-granules, and released along with insulin and C-peptide. Analysis of its gene (located on chromosome 12) supports an evolutionary relationship to calcitonin and CGRP, peptides with which it shares some biological actions. Like CGRP, IAPP antagonizes the action of insulin mainly at the level of muscle glycogen synthesis, but the levels required for this effect seem to be considerably higher than reported circulating levels. No evidence for overproduction of IAPP in diabetic subjects has been found thus far, but much more work is necessary to define its normal secretory rates and clearance. Other proposed actions of IAPP include serum calcium-lowering effects and smooth muscle relaxation; the latter effect might promote the uptake of insulin into the circulation within the islets. Deposition of amyloid is species selective due to structural differences within the central part of the molecule and may be initiated intracellularly in type II diabetes by several mechanisms. No differences in the structure of IAPP or its precursor have been found in individuals with maturity-onset diabetes of the young or type II diabetes.(ABSTRACT TRUNCATED AT 250 WORDS)

Identification of a point mutation in the human insulin gene giving rise to a structurally abnormal insulin (insulin Chicago).

Both insulin gene alleles of a diabetic patient with a mutant insulin were cloned in a lambda vector and their nucleotide sequences were determined. Nucleotide sequence analysis revealed, in one allele, a C (cytidylate) to G (guanylate) transversion in the codon for phenylalanine at position 25 of the insulin B-chain. This point mutation leads to the substitution of a leucine for phenylalanine accompanied by the loss of a restriction endonuclease Mboll recognition site and the creation of a new Rsal cleavage site at this position.

Cultured hepatoma cells as a model system for studying insulin processing and biologic responsiveness.

Monolayer cultures of minimal deviation hepatoma cells (H4-II-E-C3') bound and degraded insulin specifically, the apparent Ki value for insulin inhibition of both processes being 1 x 10(-8) M, indicating that cell-bound 125I-insulin is the substrate for subsequent hormone degradation in these cells as in isolated hepatocytes.1 The time course of insulin binding to its receptor depended on hormone concentration and temperature. Degradation of insulin also depended highly on temperature, with little or no degradation occurring at less than 20 degrees C, a temperature below which a membrane-lipid phase transition may block homone translocation or uptake. The effects of various agents on the binding and degradation of 125I-insulin also were tested. Agents believed to inhibit intralysosomal degradation of various proteins also inhibited the degradation of 125I-insulin by H4 cells (chloroquine, ammonium chloride, procaine, and lidocaine); inhibitors of energy production (dinitrophenol, sodium cyanide) inhibited degradation; an agent which inhibits microtubule function (vinblastine) blocked insulin degradation; and methylamine, reported to prevent receptor aggregation,2 also interfered with insulin processing. These findings are consistent with a model for cellular insulin processing, comprising receptor binding, clustering of receptors, endocytotic uptake, intralysosomal degradation, and extracellular release of some degradation products. H4 cells were highly sensitive to insulin. The KE for a half-maximal response of hormone-stimulated incorporationof 14C-glucose into glycogen was 10(-11) M insulin, corresponding to less than 1% receptor occupancy. This response was also mimicked by concanavalin A at a concentration of 10 microgram/ml. Vinblastine and chloroquine both significantly inhibited insulin-stimulated glucose incorporation into glycogen without affecting basal levels. However, since these inhibitory effects were not relieved by addition of excess insulin, it seems unlikely that their action on glycogen synthesis was exerted only at the level of the generation of an active intermediate or degradation product from hormone-receptor complexes. The hormone-sensitive H4 cells thus provide a useful system for further studies examining the role of insulin-receptor uptake in hormone action, receptor regulation, and signal termination.

Long-term elevation of free fatty acids leads to delayed processing of proinsulin and prohormone convertases 2 and 3 in the pancreatic beta-cell line MIN6.

To explore the role of chronically elevated free fatty acids (FFAs) in the pathogenesis of the hyperproinsulinemia of type 2 diabetes, we have investigated the effect of FFAs on proinsulin processing and prohormone convertases PC2 and PC1/PC3 in MIN6 cells cultured in Dulbecco's modified Eagle's medium with or without 0.5 mmol/l FFA mixture (palmitic acid:oleic acid = 1:2). After 7 days of culture, the percent of proinsulin in FFA-exposed cells was increased (25.9 +/-0.3% intracellular and 75.4 +/- 1.2% in medium vs. 13.5 +/-0.2 and 56.2 +/- 4.1%, respectively, in control cells). The biosynthesis and secretion of proinsulin and insulin were analyzed by comparing the incorporation of [3H]Leu and [35S]Met. In pulse-chase studies, proinsulin-to-insulin conversion was inhibited, and proinsulin in the medium was increased by 50% after 3 h of chase, while insulin secretion was decreased by 50% after FFA exposure. Levels of cellular PC2 and PC3 analyzed by Western blotting were decreased by 23 and 15%, respectively. However, PC2, PC3, proinsulin, and 7B2 mRNA levels were not altered by FFA exposure. To test for an effect on the biosynthesis of PC2, PC3, proinsulin, and 7B2, a protein required for PC2 activation, MIN6 cells were labeled with [35S]Met for 10-15 min, followed by a prolonged chase. Most proPC2 was converted after 6 h of chase in control cells, but conversion was incomplete even after 6 h of chase in FFA-exposed MIN6 cells. Media from chase incubations showed that FFA-exposed cells secreted more proPC2 than controls. Similar inhibitory effects were noted on the processing of proPC3, proinsulin, and 7B2. In conclusion, prolonged exposure of beta-cells to FFAs may affect the biosynthesis and posttranslational processing of proinsulin, PC2, PC3, and 7B2, and thereby contribute to the hyperproinsulinemia of type 2 diabetes. The mechanism of inhibition of secretory granule processing by FFAs may be through changes in Ca2+ concentration, the pH in the secretory granules, and/or other factors that may influence the activation and function of the convertases.