Search

Background: Ingestion of protein concentrates or isolates increases muscle protein synthesis rates in young and older adults. There is far less information available on the anabolic response following the ingestion of dairy wholefoods, which are commonly consumed in a normal diet. Objectives: This study investigates whether ingestion of 30 g protein provided as quark increases muscle protein synthesis rates at rest and whether muscle protein synthesis rates are further increased after resistance exercise in young and older adult males. Methods: In this parallel-group intervention trial, 14 young (18–35 y) and 15 older (65–85 y) adult males ingested 30 g protein provided as quark after a single-legged bout of resistance exercise on leg press and leg extension machines. Primed, continuous intravenous L-[ring- 13C 6]-phenylalanine infusions were combined with the collection of blood and muscle tissue samples to assess postabsorptive and 4-h postprandial muscle protein synthesis rates at rest and during recovery from exercise. Data represent means ± SDs; η 2 was used to measure the effect size. Results: Plasma total amino acid and leucine concentrations increased after quark ingestion in both groups (both time: P < 0.001; η 2 > 0.8), with no differences between groups (time × group: P = 0.127 and P = 0.172, respectively; η 2−1) and older adult males (from 0.036 ± 0.011 to 0.062 ± 0.013 %·h −1), with a further increase in the exercised leg (to 0.071 ± 0.023 %·h −1 and to 0.078 ± 0.019 %·h −1, respectively; condition: P < 0.001; η 2 = 0.716), with no differences between groups (condition × group: P = 0.747; η 2 = 0.011). Conclusions: Quark ingestion increases muscle protein synthesis rates at rest with a further increase following exercise in both young and older adult males. The postprandial muscle protein synthetic response following quark ingestion does not differ between healthy young and older adult males when an ample amount of protein is ingested. This trial was registered at the Dutch Trial register, which is accessible via trialsearch.who.int www.trialregister.nl as NL8403.

Rationale: Dietary protein may attenuate the muscle atrophy experienced by patients in the ICU, yet protein handling is poorly understood. Objectives: To quantify protein digestion and amino acid absorption and fasting and postprandial myofibrillar protein synthesis during critical illness. Methods: Fifteen mechanically ventilated adults (12 male; aged 50 ± 17 yr; body mass index, 27 ± 5 kg⋅m-2) and 10 healthy control subjects (6 male; 54 ± 23 yr; body mass index, 27 ± 4 kg⋅m-2) received a primed intravenous L-[ring-2H5]-phenylalanine, L-[3,5-2H2]-tyrosine, and L-[1-13C]-leucine infusion over 9.5 hours and a duodenal bolus of intrinsically labeled (L-[1-13C]-phenylalanine and L-[1-13C]-leucine) intact milk protein (20 g protein) over 60 minutes. Arterial blood and muscle samples were taken at baseline (fasting) and for 6 hours following duodenal protein administration. Data are mean ± SD, analyzed with two-way repeated measures ANOVA and independent samples t test. Measurements and Main Results: Fasting myofibrillar protein synthesis rates did not differ between ICU patients and healthy control subjects (0.023 ± 0.013% h-1 vs. 0.034 ± 0.016% h-1; P = 0.077). After protein administration, plasma amino acid availability did not differ between groups (ICU patients, 54.2 ± 9.1%, vs. healthy control subjects, 61.8 ± 13.1%; P = 0.12), and myofibrillar protein synthesis rates increased in both groups (0.028 ± 0.010% h-1 vs. 0.043 ± 0.018% h-1; main time effect P = 0.046; P-interaction = 0.584) with lower rates in ICU patients than in healthy control subjects (main group effect P = 0.001). Incorporation of protein-derived phenylalanine into myofibrillar protein was ∼60% lower in ICU patients (0.007 ± 0.007 mol percent excess vs. 0.017 ± 0.009 mol percent excess; P = 0.007). Conclusions: The capacity for critically ill patients to use ingested protein for muscle protein synthesis is markedly blunted despite relatively normal protein digestion and amino acid absorption.

18 Päg., The plant immune system perceives a diversity of carbohydrate ligands from plant and microbial cell walls through the extracellular ectodomains (ECDs) of pattern recognition receptors (PRRs), which activate pattern-triggered immunity (PTI). Among these ligands are oligosaccharides derived from mixed-linked β-1,3/β-1,4-glucans (MLGs; e.g. β-1,4-D-(Glc)2 -β-1,3-D-Glc, MLG43) and cellulose (e.g. β-1,4-D-(Glc)3 , CEL3). The mechanisms behind carbohydrate perception in plants are poorly characterized except for fungal chitin oligosaccharides (e.g. β-1,4-d-(GlcNAc)6 , CHI6), which involve several receptor kinase proteins (RKs) with LysM-ECDs. Here, we describe the isolation and characterization of Arabidopsis thaliana mutants impaired in glycan perception (igp) that are defective in PTI activation mediated by MLG43 and CEL3, but not by CHI6. igp1-igp4 are altered in three RKs - AT1G56145 (IGP1), AT1G56130 (IGP2/IGP3) and AT1G56140 (IGP4) - with leucine-rich-repeat (LRR) and malectin (MAL) domains in their ECDs. igp1 harbors point mutation E906K and igp2 and igp3 harbor point mutation G773E in their kinase domains, whereas igp4 is a T-DNA insertional loss-of-function mutant. Notably, isothermal titration calorimetry (ITC) assays with purified ECD-RKs of IGP1 and IGP3 showed that IGP1 binds with high affinity to CEL3 (with dissociation constant KD = 1.19 ± 0.03 μm) and cellopentaose (KD = 1.40 ± 0.01 μM), but not to MLG43, supporting its function as a plant PRR for cellulose-derived oligosaccharides. Our data suggest that these LRR-MAL RKs are components of a recognition mechanism for both cellulose- and MLG-derived oligosaccharide perception and downstream PTI activation in Arabidopsis., This work was supported by grant RTI2018-096975-B-I00 from the Spanish Ministry of Science, Innovation and Universities to AM and grant PID-2021-126006OB-100 from the Spanish Ministry of Science and Innovation to AM. This work has also been financially supported by the ‘Severo Ochoa (SO) Programme for Centres of Excellence in R&D’ from the Agencia Estatal de Investigación (AEI) of Spain (grants SEV-2016-0672 (2017-2021) and CEX2020-000999-S (2022-2025) to the CBGP). In the frame of the SO program, HM and PF-C were supported with postdoctoral fellowships. MM-D, DJB and DR were recipients of PhD Fellows PRE2019-088120 and PRE2019-091276 (SEV-2016-0672) from AEI, and IND2017/BIO-7800 from Madrid Regional Government, respectively. Research in the lab of JS was financially supported by the University of Lausanne, the European Research Council (ERC) (grant agreement no. 716358) and the Swiss National Science Foundation (grant no. 310030_204526)., With funding from the Spanish government through the ‘Severo Ochoa Centre of Excellence’ accreditation (CEX2020‐000999‐S)

Significance Although efforts have been made to determine the structure of talin and the way it interacts with integrins through the “head” domain, our work shows now that many of the previous mechanistic models based on the talin adapter are likely to be misleading as they are constructed on a crystal structure representing an improperly folded talin head domain. In this work, we identified the problem with the current talin head model and proposed a FERM-folded talin head. By analyzing these structural features of the FERM-folded talin head in a cellular context, involving also the kindlin adapter, we are making a critical and unprecedented contribution to the understanding and regulation of cell-matrix adhesions., Binding of the intracellular adapter proteins talin and its cofactor, kindlin, to the integrin receptors induces integrin activation and clustering. These processes are essential for cell adhesion, migration, and organ development. Although the talin head, the integrin-binding segment in talin, possesses a typical FERM-domain sequence, a truncated form has been crystallized in an unexpected, elongated form. This form, however, lacks a C-terminal fragment and possesses reduced β3-integrin binding. Here, we present a crystal structure of a full-length talin head in complex with the β3-integrin tail. The structure reveals a compact FERM-like conformation and a tightly associated N-P-L-Y motif of β3-integrin. A critical C-terminal poly-lysine motif mediates FERM interdomain contacts and assures the tight association with the β3-integrin cytoplasmic segment. Removal of the poly-lysine motif or disrupting the FERM-folded configuration of the talin head significantly impairs integrin activation and clustering. Therefore, structural characterization of the FERM-folded active talin head provides fundamental understanding of the regulatory mechanism of integrin function.

Proinsulin is converted into insulin by the action of two endoproteases. Type I (PC1/PC3) is thought to cleave between the B-chain and the connecting peptide (C-peptide) and type II (PC2) between the C-peptide and the A-chain. An acidic region immediately C-terminal to the point of cleavage at the B-chain/C-peptide junction is well conserved throughout evolution and has been suggested to be important for proinsulin conversion [Gross, Villa-Komaroff, Kahn, Weir and Halban (1989) J. Biol. Chem. 264, 21486-21490]. We have here compared the precise role of this region as a whole and just the first acidic residue C-terminal to the point of cleavage in processing of proinsulin by PC3. To this end, several mutations were introduced in this region of human proinsulin (native sequence, B-chain RREAEDL C-peptide): RRPAEDL (C1Pro mutant); RRLAEDL (C1Leu mutant); RRL (C1-C4del mutant); RRE (del-C1Glu mutant). Mutant and native cDNAs were stably transfected into AtT20 (pituitary corticotroph) cells, in which PC3 is known to be the major conversion endoprotease, and kinetics of proinsulin conversion were studied (pulse-chase/HPLC analysis of proinsulin-related peptides). The results show that the acidic region following the B-chain/C-peptide junction is indeed important for PC3 cleavage at this site, and that the reduced cleavage observed for the C1-C4del mutant proinsulin can be partially overcome by replacing the acidic region with a single acidic residue (del-C1Glu mutant). Replacing only the first residue of the acidic region with leucine (C1Leu mutant) has no impact on conversion, whereas its replacement with proline (C1Pro mutant) almost completely abolishes cleavage at the B-chain/C-peptide junction without affecting that at the C-peptide/A-chain junction.

Proteoglycans metabolically labelled with [35S]sulphate and [3H]glucosamine or [3H]leucine were isolated from the incubation medium and cell layer of human adult mesangial cells and glomerular visceral epithelial cells using sequential DEAE chromatography purification steps followed by gel-filtration chromatography. The proteoglycan composition of each peak was analysed by treatment with HNO2, chondroitinase ABC or chondroitinase AC followed by chromatography on Sephadex G-50 columns. Heparan sulphate proteoglycan (HSPG) and dermatan sulphate proteoglycan were detected in both the culture medium and cell layer of mesangial cells. Culture medium of glomerular visceral epithelial cells contained HSPG and a second proteoglycan with the properties of a hybrid molecule containing HS and chondroitin sulphate (CS). The cell layer contained HSPG and CSPG. Detailed analysis of the hybrid molecule revealed that it had an apparent molecular mass of 400 kDa. SDS/PAGE of hybrid molecules, after treatment with heparitinase and chondroitinase ABC, revealed a core protein of 80 kDa. Using 1.8% polyacrylamide/0.6% agarose-gel electrophoresis, we deduced that the HS and CS were independently attached to one core protein. Because glomerular-basement-membrane HSPG is thought to be derived from mesangial cells and glomerular visceral epithelial cells and this molecule is involved in several kidney diseases, we investigated its synthesis in more detail. Anti-(rat glomerular-basement-membrane HSPG) monoclonal antibodies (JM403) and anti-(human glomerular-basement-membrane HSPG) polyclonal antibodies (both antibodies known to react with the large basement-membrane HSPG, perlecan) reacted strongly with HSPG obtained from both mesangial cells and glomerular visceral epithelial cells. However, the hybrid molecule did not react with these antibodies, suggesting that the HS side chain and the core protein were different from glomerular-basement-membrane HSPG. To quantify HS we performed an inhibition ELISA using mouse antibodies specific for glomerular-basement-membrane HS glycosaminoglycan side chains. Glomerular visceral epithelial cells produced significantly higher levels of HS (between 197.56 and 269.40 micrograms/72 h per 10(6) cells) than mesangial cells (between 29.8 and 45.5 micrograms/72 h per 10(6) cells) (three different cell lines; n = 3; P < 0.001). HS production by these cells was inhibited by cycloheximide, revealing that it was synthesized de novo. Expression of perlecan mRNA, demonstrated using reverse transcriptase PCR, was different in the two cell types. We conclude that glomerular visceral epithelial cells and mesangial cells have characteristic patterns of proteoglycan production. Glomerular visceral epithelial cells produced a hybrid proteoglycan containing CS and HS independently attached to its core protein.(ABSTRACT TRUNCATED AT 250 WORDS)

A modified procedure for determining the pattern of peptidoglycan synthesis during the division cycle has allowed the measurement of the rate of side wall synthesis during the division cycle without the contribution due to pole formation. As predicted by a model proposing that the surface growth of the cell is regulated by mass increase, we find a decrease in side wall synthesis in the latter half of the division cycle. This supports the proposal that, upon invagination, pole growth accommodates a significant proportion of the increasing cell mass and that residual side wall growth occurs in response to the residual mass increase not accommodated by pole volume. The observed side wall synthesis patterns support the proposal that mass increase is a major, and possibly sole, regulator of bacterial surface increase. Membrane synthesis during the division cycle of the gram-negative, rod-shaped bacteria Escherichia coli and Salmonella typhimurium has also been measured with similar methods. The rate of membrane synthesis--measured by incorporation of radioactive glycerol or palmitate relative to simultaneous labeling with radioactive leucine--exhibits the same pattern as peptidoglycan synthesis. The results are compatible with a model of cell surface growth containing the following elements. (i) During the period of the division cycle prior to invagination, growth of the cell occurs predominantly in the side wall and the cell grows only in length. (ii) When invagination begins, pole growth accommodates some cytoplasmic increase, leading to a concomitant decrease in side wall synthesis. (iii) Surface synthesis increases relative to mass synthesis during the last part of the division cycle because of pole formation. It is proposed here that membrane synthesis passively follows the pattern of peptidoglycan synthesis during the division cycle.

Stable isotope labeling of amino acids in cell culture was used for Bifidobacterium longum. A comprehensive proteomic strategy was developed and validated by designing an appropriate semidefined medium that allows stable replacement of natural leucine by [13C6]leucine. Using this strategy, proteins having variations of at least 50% in their expression rates can be quantified with great confidence, This work was financed by European Union FEDER funds and the Spanish Plan Nacional de I + D (project AGL2004-06727-C02-01/ALI and fellowship PR2006-0421) and by the Swiss National Science Foundation (grant 3100-A0-104214).

Mutations in the hepatocyte nuclear factor-1alpha (HNF-1alpha) have been linked to subtype 3 of maturity-onset diabetes of the young (MODY3), which is characterized by a primary defect in insulin secretion. The role of HNF-1alpha in the regulation of pancreatic beta-cell function was investigated. Gene manipulation allowed graded overexpression of HNF-1alpha and controlled dominant-negative suppression of HNF-1alpha function in insulinoma INS-1 cells. We show that HNF-1alpha is essential for insulin gene transcription, as demonstrated by a pronounced decrease in insulin mRNA expression and in insulin promoter activity under dominant-negative conditions. The expression of genes involved in glucose transport and metabolism including glucose transporter-2 and L-type pyruvate kinase is also regulated by HNF-1alpha. Loss of HNF-1alpha function leads to severe defects in insulin secretory responses to glucose and leucine, resulting from impaired glucose utilization and mitochondrial oxidation. The nutrient-evoked ATP production and subsequent changes in plasma membrane potential and intracellular Ca2+ were diminished by suppression of HNF-1alpha function. These results suggest that HNF-1alpha function is essential for maintaining insulin storage and nutrient-evoked release. The defective mitochondrial oxidation of metabolic substrates causes impaired insulin secretion, indicating a molecular basis for the diabetic phenotype of MODY3 patients.

Proteoglycans metabolically labelled with [35S]sulphate and [3H]glucosamine or [3H]leucine were isolated from the incubation medium and cell layer of human adult mesangial cells and glomerular visceral epithelial cells using sequential DEAE chromatography purification steps followed by gel-filtration chromatography. The proteoglycan composition of each peak was analysed by treatment with HNO2, chondroitinase ABC or chondroitinase AC followed by chromatography on Sephadex G-50 columns. Heparan sulphate proteoglycan (HSPG) and dermatan sulphate proteoglycan were detected in both the culture medium and cell layer of mesangial cells. Culture medium of glomerular visceral epithelial cells contained HSPG and a second proteoglycan with the properties of a hybrid molecule containing HS and chondroitin sulphate (CS). The cell layer contained HSPG and CSPG. Detailed analysis of the hybrid molecule revealed that it had an apparent molecular mass of 400 kDa. SDS/PAGE of hybrid molecules, after treatment with heparitinase and chondroitinase ABC, revealed a core protein of 80 kDa. Using 1.8% polyacrylamide/0.6% agarose-gel electrophoresis, we deduced that the HS and CS were independently attached to one core protein. Because glomerular-basement-membrane HSPG is thought to be derived from mesangial cells and glomerular visceral epithelial cells and this molecule is involved in several kidney diseases, we investigated its synthesis in more detail. Anti-(rat glomerular-basement-membrane HSPG) monoclonal antibodies (JM403) and anti-(human glomerular-basement-membrane HSPG) polyclonal antibodies (both antibodies known to react with the large basement-membrane HSPG, perlecan) reacted strongly with HSPG obtained from both mesangial cells and glomerular visceral epithelial cells. However, the hybrid molecule did not react with these antibodies, suggesting that the HS side chain and the core protein were different from glomerular-basement-membrane HSPG. To quantify HS we performed an inhibition ELISA using mouse antibodies specific for glomerular-basement-membrane HS glycosaminoglycan side chains. Glomerular visceral epithelial cells produced significantly higher levels of HS (between 197.56 and 269.40 micrograms/72 h per 10(6) cells) than mesangial cells (between 29.8 and 45.5 micrograms/72 h per 10(6) cells) (three different cell lines; n = 3; P < 0.001). HS production by these cells was inhibited by cycloheximide, revealing that it was synthesized de novo. Expression of perlecan mRNA, demonstrated using reverse transcriptase PCR, was different in the two cell types. We conclude that glomerular visceral epithelial cells and mesangial cells have characteristic patterns of proteoglycan production. Glomerular visceral epithelial cells produced a hybrid proteoglycan containing CS and HS independently attached to its core protein.(ABSTRACT TRUNCATED AT 250 WORDS)

We have investigated how neurons in the optic tecta of embryonic day 16 chick embryos depend for survival on their afferents from the retina. To distinguish between activity-mediated effects and other, “trophic,” ones, we compared the effects on the tectal neurons of blocking intraocular axoplasmic transport (with colchicine) or action potentials (by means of TTX). Both interventions rapidly induced the appearance of dying (pyknotic) neurons in the tectum, with major increases in their number occurring within 13 hr post-colchicine and within 9 hr post-TTX. Following both drugs, the dying neurons were morphologically similar, and in both cases the cell death depended on protein synthesis. However, the effects of colchicine and of TTX could be dissociated, since the most superficial tectal neurons became pyknotic only in response to colchicine, and, with a sufficiently short survival time (9 hr), the deep cells of the stratum griseum centrale became pyknotic only in response to TTX. We hence argue that the survival of the tectal neurons depends on their ongoing maintenance by substances released from retinotectal axon terminals, the release being activity dependent in the case of the deep neurons but independent of activity in the case of the superficial ones.

Rat pancreatic islets were exposed to a combination of analogs of arginine (3 mM canavanine) and lysine (3 mM thialysine) for 2 h and then labeled with [3H]leucine in the continued presence of the analogs. Control islets were incubated in parallel without analogs. Prelabeled islets were then incubated for a 3-h chase period without analogs. Incorporation of the analogs blocked conversion of newly synthesized, radioactive, proinsulin to insulin. No untoward toxic effects of the analogs were found on nonradioactive insulin as measured by radioimmunoassay. The rate of release of the modified proinsulin was no different from that found for proinsulin newly synthesized in the absence of analogs and, as such, susceptible to the normal action of the enzymes responsible for proinsulin to insulin conversion. These results confirm the previously untested hypothesis that the beta-granule is the minimal functional unit of release; granules are thus handled by the B-cell and released at a rate which is independent of the physicochemical nature of their contents.

Rat islets were maintained for 3 days in tissue culture at 2.8, 8.3, or 16.7 mM glucose, in the presence of [3H]leucine to label insulin stores. The immunoreactive insulin content of islets after 3 days at either 2.8 or 16.7 mM glucose was lower than that of islets exposed to 8.3 mM glucose. The specific radioactivity of stored labeled insulin, however, increased with increasing glucose. Islet insulin turnover was thus highest at 16.7 mM glucose. During a subsequent 1-day chase period at 2.8, 8.3, or 16.7 mM glucose for each of the three groups of islets prelabeled at these same three concentrations, all islets responded to glucose with increased insulin release. Release of immunoreactive insulin was highest at all three chase glucose concentrations for those islets preexposed for 3 days to 16.7 mM glucose. After 3-day labeling of islets at 8.3 mM glucose, there was an inverse relationship between the rate of release of labeled insulin and its intracellular degradation in response to the three glucose concentrations during the chase period. Those islets prelabeled at either 2.8 or 16.7 mM glucose showed much lower rates of intracellular degradation of labeled insulin stores during the chase period regardless of the ambient glucose or of the rate of release of the labeled hormone. Adaptive changes in islet insulin handling thus occurred during the 3-day maintenance period at different glucose concentrations. Whereas glucose concentration is, of course, thought to regulate insulin biosynthesis and release directly, regulation of degradation of stored insulin within B-cells appears to reflect both the relative activity of biosynthesis and release of the hormone and the insulin content of the islets. This last and striking relation is best interpreted as reflecting two labile, exchangeable, granule pools, one preferentially available for release and the other for intracellular degradation.

Effects of high glucose on the production of heparan sulfate proteoglycan by mesangial and epithelial cells. Changes in heparan sulfate metabolism may be important in the pathogenesis of diabetic nephropathy. Recent studies performed on renal biopsies from patients with diabetic nephropathy revealed a decrease in heparan sulfate glycosaminoglycan staining in the glomerular basement membrane without changes in staining for heparan sulfate proteoglycan-core protein. To understand this phenomenon at the cellular level, we investigated the effect of high glucose conditions on the synthesis of heparan sulfate proteoglycan by glomerular cells in vitro. Human adult mesangial and glomerular visceral epithelial cells were cultured under normal (5mM) and high glucose (25mM) conditions. Immunofluorescence performed on cells cultured in 25mM glucose confirmed and extended the in vivo histological observations. Using metabolic labeling we observed an altered proteoglycan production under high glucose conditions, with predominantly a decrease in heparan sulfate compared to dermatan sulfate or chondroitin sulfate proteoglycan. N-sulfation analysis of heparan sulfate proteoglycan produced under high glucose conditions revealed less di- and tetrasaccharides compared to larger oligosaccharides, indicating an altered sulfation pattern. Furthermore, with quantification of glomerular basement membrane heparan sulfate by ELISA, a significant decrease was observed when mesangial and visceral epithelial cells were cultured in high glucose conditions. We conclude that high glucose concentration induces a significant alteration of heparan sulfate production by mesangial cells and visceral epithelial cells. Changes in sulfation and changes in absolute quantities are both observed and may explain the earlier in vivo observations. These changes may be of importance for the altered integrity of the glomerular charge-dependent filtration barrier and growth-factor matrix interactions in diabetic nephropathy.

Rat pancreatic islets were maintained in tissue culture (8.3 mM glucose) for 3 days in the presence of [3H]leucine in order to label insulin stores. Alternatively, islets were labeled for 1 h at the end of a 3-day maintenance period in order to label only newly synthesized insulin. During a 24-h chase incubation, 13% of the prelabeled insulin stores (3-day label) had been released. By contrast, 30% of newly synthesized (1-h label) insulin was released during a similar chase period. Since islets were handled identically aside from the respective time of exposure to [3H]leucine, the total immunoreactive islet insulin content and that released into the medium was similar for both groups of islets. The specific radioactivity of the labeled insulin in the medium at the end of the chase period was thus lower than that remaining in the islets for islets prelabeled for 3 days but was higher after the labeling of newly synthesized insulin for 1 h. When the rate of newly synthesized insulin was followed during a 4-...

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.

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.