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. Author manuscript; available in PMC: 2018 Apr 23.
Published in final edited form as: Diabetologia. 2016 Jan 30;59(7):1480–1491. doi: 10.1007/s00125-016-3868-9

Autophagy is a major regulator of β-cell insulin homeostasis

Yael Riahi 1,*, Jakob D Wikstrom 1,5,*, Etty Bachar-Wikstrom 1, Nava Polin 1, Hava Zucker 1, Myung-Shik Lee 2, Wenying Quan 3, Leena Haataja 4, Ming Liu 4, Peter Arvan 4, Erol Cerasi 1, Gil Leibowitz 1
PMCID: PMC5912938  NIHMSID: NIHMS959110  PMID: 26831301

Abstract

Aims/hypothesis

We studied the role of protein degradation pathways in the regulation of insulin production and secretion and hypothesized that autophagy regulates proinsulin degradation, thereby modulating β-cell function.

Methods

Proinsulin localization in autophagosomes was demonstrated by confocal and electron microscopy. Autophagy was inhibited by knockdown of autophagy genes and using the H+-ATPase inhibitor bafilomycin A1. Proinsulin and insulin content and secretion were assessed in static incubations by ELISA and RIA.

Results

Confocal and electron microscopy showed proinsulin localized in autophagosomes and lysosomes, suggesting proinsulin is delivered to autophagosomes at the trans-Golgi. βAtg7 knockout mice had proinsulin-containing P62/SQSTM1+ aggregates in β-cells, indicating proinsulin is regulated by autophagy in vivo. Short-term bafilomycin A1 treatment and Atg5/Atg7 knockdown increased steady-state proinsulin and hormone precursor chromogranin A content. Atg5/7 knockdown also increased glucose- and non-fuel-stimulated insulin secretion. Finally, proinsulin mutants that are irreparably misfolded and trapped in the ER are more resistant to degradation by autophagy.

Conclusions/interpretation

In the β-cell, transport-competent secretory peptide precursors including proinsulin are regulated by autophagy, whereas for transport-incompetent proinsulin mutants efficient clearance by alternative degradative pathways may be necessary to avoid β-cell proteotoxicity. Reduction of autophagic degradation of proinsulin increases its residency in the secretory pathway, followed by enhanced secretion in response to stimuli.

Keywords: proinsulin, insulin secretion, β-cells, autophagy, lysosome – proteasome, type 2 diabetes, protein degradation

Orci et al showed three decades ago that β-cell insulin stores are regulated by crinophagy, where granules fuse directly with large vacuolar lysosomes to generate crinophagic bodies, where granule contents are degraded [1]. Insulin may also reach lysosomes via autophagosomes that engulf cytosolic components containing secretory granules (macroautophagy), or by lysosomal engulfment of a single granule (microautophagy). It was recently reported that in contrast to most mammalian cells, in β-cells, starvation induced lysosomal degradation of nascent insulin granules triggering lysosomal recruitment and activation of mTORC1, thereby suppressing autophagy [2]. This would reduce insulin secretion in fasting; indeed, stimulation of autophagy enhanced secretion in starved β-cells. Starvation is an extreme situation; therefore the relevance of these findings for β-cell physiology is unclear.

The stimulated β-cell confronts a high protein burden due to proinsulin biosynthesis rates reaching 106 molecules/min, ~50% of total protein synthesis [3]. Proinsulin rapidly enters the ER and undergoes oxidative folding. Misfolded proinsulin may be degraded via a process called ER-associated degradation (ERAD), which includes translocation to the cytosolic side of the ER membrane, followed by ubiquitination and delivery to proteasomes for degradation (4). Properly folded proinsulin is transported to the cis-Golgi, then moving forward between multiple cisternae to the trans-Golgi network (TGN), where cisternae disintegrate to produce carriers that convey proteins to the plasma membrane, secretory granules, endosomes and lysosomes [5].

Autophagy directs cytoplasmic components to lysosomes for degradation [6]. Double-membrane vesicles (autophagosomes) sequester cytosolic constituents including misfolded proteins and damaged organelles, then fuse with lysosomes where the material is degraded and recycled. This pathway is tightly regulated by multiple proteins encoded by autophagy genes (ATGs) [7–9]. Transgenic mice with β-cell Atg7 deficiency exhibit islet degeneration, impaired insulin secretion and glucose intolerance, indicating that autophagy is essential for β-cell wellbeing [10; 11]. Alterations of autophagy have been described in animal models of diabetes and in β-cells of type 2 diabetic patients [12; 13]. Beyond ubiquitous roles, autophagy also regulates tissue-specific functions [12]. Herein we studied the regulation of β-cell insulin-like peptides (ILP) by autophagy and its effects on insulin secretion.

Methods

Islet isolation, cell culture and reagents

Pancreata were obtained from 16-week-old β-cell Atg7−/− mice; fed blood glucose levels were ~12 mmol/l [14]. Islets were isolated from C57BL6 mice by collagenase injection to the bile duct. Animal use was approved by the Institutional- Animal Care and Use Committee of the Hebrew University-Hadassah Medical Organization. INS-1E and BON-1 cell lines were grown as previously described [15–16]. Bafilomycin-A1, MG132, lactacystin, brefeldin-A, trehalose, tat-Beclin1 and diazoxide were obtained from Sigma (Rehovot, Israel).

RNAi knockdown

INS-1E cells were transfected with siRNA or scrambled siRNA using Jetprime transfection reagent (Polyplus Transfection, Illkirch-Graffenstaden, France). Specific anti-rat Atg5 and Atg7 siRNA smartpool sequences were ordered from Dharmacon (Lafayette, LA). Cells were harvested 48-h following transfection.

Immunofluorescence

Paraffin sections were rehydrated and antigens retrieved. The following antibodies were used: guinea pig anti-insulin 1:200 (Dako, Glostrup, Denmark), mouse anti-proinsulin 1:200 (Developmental Studies Hybridoma Bank, Iowa City, IA) and rabbit anti-P62/SQSTM1 1:200 (Cell Signaling Technology, MA). Secondary antibodies were all from Jackson ImmunoResearch Laboratories (West Grove, PA).

Live cell imaging

A Zeiss LSM-710 confocal microscope with an incubator (37°C and 5% CO2) was used (Carl Zeiss, Oberkochen, Germany). Cells were seeded on 8-chamber borosilicate coverglass plates (Nunc, Roskilde, Denmark) and transfected the subsequent day with the following constructs using Lipofectamine-2000 and Optimem (Life Technologies, Carlsblad, CA): LAMP1-eGFP [17], LC3-mCherry [17], P62/SQSTM1-mCherry [18], B4GALT1-mTurqoise2 (mTurqoise-Golgi) (plasmid #36205; Addgene) [19], WT proinsulin-eGFP, WT proinsulin-mCherry, Akita proinsulin-eGFP [20] and G(B23)V proinsulin-eGFP [21]. eGFP was excited using a 488nm argon laser and mCherry with a 561nm helium/neon laser. Image processing was with Metamorph software (Molecular Devices, Sunnyvale, CA).

Immunogold electron microscopy (EM)

Mouse pancreata were fixed using cold 2.5% glutaraldehyde, 2% formaldehyde and 0.1mol/l acodylic acid, and cut into ultra-thin sections. These were transferred to formvar-coated 200 mesh nickel grids. Sections were treated with conditioning medium for 5-min to block non-specific binding followed by 2-h incubation with primary polyclonal guinea pig anti-mouse (pro)insulin (1:200, Dako) and secondary donkey anti-guinea pig gold conjugate (Jackson ImmunoResearch). Grids were washed in PBS-glycine, stained with neutral uranyl acetate oxalate for 5-min, then with 2% uranyl acetate in H2O for 10-min. The grids were then embedded in 2% methyl cellulose/uranyl acetate and imaged by transmission EM (Tecnai 12 TEM, Phillips).

Western blotting

Protein levels were assessed using antibodies against: ATG5, ATG7, LC3-I/II (Cell Signaling), HSP90 (BD Biosciences, San Jose, CA), eGFP, GAPDH, and Chromogranin-A (CgA) (Abcam, Cambridge, UK). Peroxidase-conjugated AffiniPure anti-rabbit, anti-mouse and anti-goat IgG from Jackson Immunoresearch Laboratories were used as secondary antibodies.

Insulin, proinsulin and chromogranin-A content and secretion

Insulin response to secretagogues was evaluated by static incubation. Cells were pre-incubated for 60 min in RPMI-1640 containing 3.3mmol/l glucose, then consecutively incubated at 3.3mmol/l and 16.7mmol/l glucose or 30mmol/l KCl or 10μmol/l glyburide for 1 h at 37°C in 1ml modified Krebs-Ringer bicarbonate buffer containing 20mmol/l HEPES and 0.25% BSA (KRBH-BSA). Medium was collected, centrifuged, and frozen at -20°C. Insulin and proinsulin were extracted from INS-1E cells and islets in 0.1% BSA-GB/NP-40 solution and determined by RIA and ELISA. Islet experiments were performed in batches of 15–35 mouse or human islets/chamber in four-chamber culture plates (Nunclon δ Multidishes; Nunc) in triplicates or quadruplicates. CgA secretion was measured following 1 h incubation of BON-1 cells in KRH medium (Hepes 25mM in NaOH pH 7.4; NaCl 120mM, KCl 5mM; MgSO4 1.2mM; CaCl2 1.3mM; KH2PO4 1.3mM; glucose 17.5mM). Insulin immunoreactivity in extracts and medium of INS-1E cells and mouse islets was determined using a rat RIA kit (Millipore, Darmstadt, Germany); this antibody cross-reacts with proinsulin and its conversion intermediates; therefore we refer to it as total ILP, rather than insulin. The ADVIA Centaur chemiluminesence insulin assay (Siemens Healthcare Diagnostics, Malvern, PA) was used for measuring human islet insulin. The anti-serum for human insulin does not cross-react with proinsulin. Proinsulin content in INS-1E cells and mouse and human islets was determined using specified ELISA kits (Mercodia, Uppsala, Sweden). The antiserum for proinsulin does not cross-react with insulin or C-peptide. The intra- and inter-assay CVs of the proinsulin assays were <3.6 and <6.3%, respectively. Human CgA was analyzed by ELISA (Cisbio Bioassays, Bedford, MA).

Oxygen consumption

Oxygen consumption was measured in INS-1E cells in V7 plates in a XF24 respirometer (Seahorse Bioscience, Billerica, MA) as described [22]. Measurements were recorded before and consecutively after stimulation with 20mmol/l glucose, followed by 1μM FCCP and finally rotenone and antimycin (both at 50μmol/l) (Sigma).

Statistical analysis

Data are means +/− SEM. Differences between multiple groups were analyzed by one-way ANOVA with post-hoc Sidak or Bonferroni corrected two-tailed t-test. Two-tailed paired Student’s t-test was used to compare differences between two groups, unless otherwise indicated. One-sample Student’s t test was used to validate statistical differences in experiments expressing data as relative of control. p<0.05 was considered significant.

Results

Proinsulin degradation by lysosomes

INS-1E β-cells and mouse or human islets were incubated for 1–2 h with bafilomycin-A1, an established vacuolar H+-ATPase inhibitor. Surprisingly, short-term (≤120 min) treatment with 100nmol/l bafilomycin-A1 increased the steady-state proinsulin content both in INS-1E cells and islets (Figure 1A). In mouse islets, proinsulin was increased by ~70%, accompanied by increased total ILP content. In INS-1E cells and human islets there was a smaller increase of proinsulin, without affecting insulin content (Figure 1A). Proinsulin increase after short-term bafilomycin-A1 treatment was unexpected considering its abundance in β-cells, suggesting that substantial amounts of proinsulin are degraded in lysosomes. This cannot be explained by inhibition of secretion, as 2-h treatment of islets with bafilomycin-A1 at 11.1mmol/l glucose did not decrease insulin or proinsulin secretion (Supplemental Figure 1). Furthermore, proinsulin secretion inhibition by diazoxide failed to increase islet proinsulin (Supplemental Figure 1). In mouse islets, bafilomycin-A1 similarly increased proinsulin content at 5.5–22 mmol/l glucose, suggesting that lysosomal degradation of proinsulin is not affected by ambient glucose (Supplemental Figure 2). Bafilomycin-A1 increased both proinsulin and insulin; thus the effect on proinsulin content cannot be explained by perturbation of proinsulin conversion to insulin.

Figure 1.

Figure 1

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(A) INS-1E cells and islets were treated for 1 h (islets) and 2 h (INS-1E cells) at 11.1mmol/l glucose with or without bafilomycin-A1 (100nmol/l) followed by islet extraction and measurement of proinsulin by ELISA and total ILP by RIA. Human islets were incubated for 1-h in CMRL-1066 medium at 5.5mmol/l glucose with or without bafilomycin-A1 followed by islet extraction and measurement of proinsulin and ILP (INS-1E: 6 independent experiments in triplicates; mouse islets: 4–6 independent experiments in quadruplicates). (B–C) INS-1E cells were transfected with proinsulin-eGFP construct, then treated for 2 or 4 h with bafilomycin-A1 (100nmol/l) or lactacystin (10μmol/l) or MG132 (50μmol/l). Proinsulin level analyzed by Western blotting for eGFP (n=3–4 at 2 h and n=5–7 at 4 h). (D–E) INS-1E cells were incubated with cycloheximide for 4 h with or without bafilomycin-A1 or lactacystin. Proinsulin-eGFP content assessed by Western blotting. A representative experiment (D) and quantification (E) are shown (n=4). * p<0.05, ** p<0.01.

To gain mechanistic information, we used proinsulin constructs tagged with various fluorescent markers. eGFP or mCherry was inserted into the C-peptide domain of proinsulin [20]. This enables monitoring proinsulin and C-peptide levels, without interference of fully-processed insulin, devoid of tag. We studied the effects of bafilomycin-A1 and proteasome inhibitors (MG-132 and lactacystin) on proinsulin level and degradation in INS-1E cells (Figure 1B–E). Bafilomycin-A1 increased the proinsulin-eGFP level, thus adequately reflecting changes in proinsulin concentrations measured by ELISA. Proteasome inhibitors had a small effect on proinsulin in INS-1E cells, consistently smaller than that of bafilomycin-A1 (Figure 1B–E). Treatment of islets with lactacystin for 2-h was associated with decreased, rather than increased proinsulin content (Supplemental Figure 3), further suggesting that lysosomes are the main regulators of proinsulin degradation. To exclude interference with changes in translation rate, β-cells were treated with the protein biosynthesis inhibitor cycloheximide; bafilomycin-A1 again increased proinsulin at 2-h and 4-h, while there was a small, non-significant effect of lactacystin (Figure 1D–E). Bafilomycin-A1 may impair acidification of young secretory granules, where proinsulin is processed to insulin; this may inhibit proinsulin-to-insulin conversion, thereby increasing proinsulin content. To exclude this, we analyzed the bafilomycin-A1 effect on C-peptide-eGFP expression in INS-1E cells treated with cycloheximide (Figure 1D–E and Supplemental Figure 4). Bafilomycin-A1 induced a parallel increase in proinsulin-eGFP and C-peptide-eGFP, indicating that proinsulin conversion to insulin was not impaired.

Collectively, these findings indicate that the bafilomycin-A1 effect on β-cell proinsulin is mediated via inhibition of protein degradation, rather than impairment of proinsulin processing or insulin secretion, and that lysosomal degradation of proinsulin dominates over that by the proteasome.

Lysosomal degradation of proinsulin is via autophagy

Proteins may reach the lysosome through multiple pathways including, but not limited to, autophagy. We have previously shown that in β-cell lines and islets autophagic flux is rapid, evident by high LC3-II turnover [23]. Consistently, bafilomycin-A1, but not the proteasome inhibitor, robustly increased LC3-II, along with proinsulin-eGFP (and C-peptide-eGFP) expression (Supplemental Figure 5). To clarify whether proinsulin is delivered to lysosomes via autophagy, we performed imaging studies and knocked down autophagy genes. INS-1E cells were co-transfected with mCherry- or eGFP-labeled proinsulin and the (auto)lysosome markers LAMP-1-eGFP or P62/SQSTM1-mCherry and then treated with or without bafilomycin-A1 (Figure 2A–B). At baseline, proinsulin-mCherry and LAMP-1-eGFP colocalization was relatively scant. Inhibition of lysosome activity resulted in accumulation of proinsulin-mCherry in lysosomes already at 30-min, suggesting that proinsulin turnover is rapid.

Figure 2.

Figure 2

Figure 2

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(A–B) INS-1E β-cells were transfected with wild-type proinsulin (PI) labeled with mCherry or eGFP and with the autolysosome markers LAMP-1-eGFP or with P62/SQSTM1-mCherry. After 24 h, cells were treated with bafilomycin-A1 for different times, followed by confocal microscope imaging. (B) Quantification of proinsulin-P62/SQSTM1 colocalization (n=3). (C–D) INS-1ECells were transfected with proinsulin-eGFP or proinsulin-mCherry and P62/SQSTM1-mCherry and then treated with 50nmol/l rapamycin for 16 h. (D) Quantification of proinsulin-P62/SQSTM1 colocalization (lower panel) (n=3). (E) Cells were incubated with cycloheximide for 2 h with or without 50nmol/l rapamycin and proinsulin content analyzed by ELISA (n=3 in triplicates). (F) INS-1E cells were treated overnight with 100mmol/l trehalose or with tat-Beclin1 for 3 h. Autophagy was assessed by Western blotting for LC3-II and P62/SQSTM1 and proinsulin content analyzed by ELISA (n=3 in triplicates). (G) INS-1E cells were transfected with proinsulin-eGFP, LC3-mCherry and B4GALT1-mTurquoise2 (mTurquoise-Golgi) constructs, then treated with and without bafilomycin A1. Proinsulin and B4GALT1 localization in autophagosomes (LC3-eGFP+ punctae) analyzed by confocal microscope. Size bar=10 μm.

We have shown that the mTORC1 inhibitor rapamycin stimulates autophagy in β-cells [23]. Rapamycin increased the localization of proinsulin-eGFP/mCherry in P62/QSTM1+ and LAMP-1+ vesicles (Figure 2C); this was associated with a small, borderline significant, decrease in proinsulin level (Figure 2D). The autophagy stimulators tat-Beclin1 and trehalose also decreased proinsulin level in INS-1E cells (Figure 2E). Altogether, these findings suggest that proinsulin is assigned for lysosomal degradation through autophagy.

Autophagosome maturation involves conversion of soluble cytosolic LC3 (LC3-I) to vesicle-associated form (LC3-II), which appears as dots (punctae) by fluorescence microscopy. Proteins destined for different cellular domains are sorted in the TGN [23]; B4GALT1 is an integral membrane protein of this compartment. β-Cells were transfected with constructs expressing LC3-mCherry, proinsulin-eGFP and B4GALT1-mTurquoise, and analyzed by confocal microscopy. LC3-mCherry colocalized with proinsulin-eGFP and B4GALT1-mTurqoise in autophagic punctae (Figure 2F), indicating that proinsulin is present in autophagosomes containing TGN-derived membrane proteins. Treatment with brefeldin-A, which prevents protein transport from ER to Golgi, abolished the lysosomal degradation of proinsulin (Supplemental Figure 6), further suggesting that proinsulin channeling for autophagic degradation occurs downstream to the ER.

ATG5 and ATG7 are required for autophagosome elongation and maturation [7]. Partial depletion of both ATGs impaired autophagy, evident by decreased LC3-II expression (Figure 3A–D). This resulted in ~50% increase of β-cell proinsulin at 3.3–22.2 mmol/l glucose, without affecting insulin content (Figure 3E–G and Supplemental Figure 7). This cannot be explained by inhibition of proinsulin secretion, as increased proinsulin content in autophagy-deficient INS-1E cells was accompanied by increased proinsulin secretion (see Figure 4B).

Figure 3.

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INS-1E cells were transfected with scrambled siRNA or with siRNA directed against Atg5 and Atg7 for 48 h. (A, B) Western blotting for ATGs (n=3). (C) Autophagy assessed by Western blotting for LC3-II in cells treated with or without bafilomycin A1; quantification of LC3- II in bafilomycin A1-treated cells is shown in (D) (n=3). (E–F) Proinsulin and total ILP measured by ELISA and RIA in control and Atg5/7-knockdown cells (n=3 in triplicates); (G) proinsulin/total ILP ratio. * p<0.05, ** p<0.01, *** p<0.001.

Figure 4.

Figure 4

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Effects of Atg5/7-knockdown on proinsulin and insulin secretion. INS-1E cells were transfected with scrambled siRNA or siRNA directed against Atg5 and Atg7 for 48 h. (A) Glucose-dependent stimulation of total ILP and proinsulin secretion. Control (squares) and Atg5/7 knockdown cells (circles) were incubated at 3.3, 11.1 and 22.2mmol/l glucose for 1 h (closed circles and squares). ILP-closed symbols; proinsulin-open symbols (n=3 in duplicates). (B–C) ILP and proinsulin secretion assessed by static incubations at 3.3mmol/l and after stimulation with 16.7mmol/l glucose (n=3 in triplicates), 30mmol/l KCl (n=3) and 10μmol/l glyburide (n=3). (B) Basal and stimulated ILP secretion; (C) stimulated proinsulin secretion; (D) proinsulin/ILP ratio; (E) Proinsulin and ILP content in cell extracts. * p<0.05, ** p<0.01, *** p<0.001.

Altogether, our findings show that proinsulin is assigned for lysosomal degradation through autophagy, and may suggest that this occurs at the TGN.

Effects of inhibiting autophagy on proinsulin and insulin secretion

Autophagy deficiency induced by Atg5/7-knockdown did not affect basal insulin secretion. By contrast, it increased glucose-stimulated insulin secretion (Figure 4A), especially at submaximal glucose (11.1mmol/l). Atg5/7-knockdown increased in parallel insulin and proinsulin secretion in response to glucose, plasma membrane depolarization by KCl and the sulfonylurea glyburide (Figure 4A–C). In autophagy-deficient cells, the relative increase in proinsulin secretion exceeded that of insulin (Figure 4B–C), suggesting that processing of rescued proinsulin to insulin was incomplete. Atg5/7-knockdown did not affect mitochondrial respiration (Supplemental Figure 8). Inhibition of autophagy increased insulin secretion by KCl and glyburide, further suggesting that its effects on secretion are not mediated via modulation of mitochondrial metabolism.

Collectively, our findings show that autophagy restrains insulin secretion; its inhibition enables proinsulin accumulation in secretory granules, followed by processing and increased secretion in response to stimuli.

Regulation of proinsulin by autophagy in vivo

The intracellular localization of ILP was studied by EM. Pancreatic sections were stained by immuno-gold labeling for insulin using an antibody that recognizes all ILP, including proinsulin. As expected, ILP were abundant in secretory granules and were not observed in non-β-cells (Supplemental Figure 9), indicating specific staining. ILP was observed in lysosomes containing single secretory granules suggestive of microautophagy, and in crinophagic bodies, containing single secretory granules (Figure 5A), as well as multigranular bodies which contain secretory granule core-like material (not shown). In addition, we observed double-membrane structures, likely autophagosomes, containing dispersed ILP, not in the form of insulin granules (Figure 5A). To further show that proinsulin is regulated by autophagy in vivo, we immuno-stained pancreatic sections of βAtg7 knockout mice for proinsulin and for P62/SQSTM1. Life-long Atg7 deficiency results in β-cell degeneration, insulin deficiency and glucose intolerance. βAtg7 knockout mice were depleted of insulin and contained P62/SQSTM1+ ubiquitinated protein aggregates [10; 11]. We found that islets were also depleted of proinsulin (Figure 5B); despite this, proinsulin was found in part of the P62/SQSTM1+ aggregates, suggesting that it serves as substrate for autophagy also in vivo.

Figure 5.

Figure 5

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(A) EM analysis of insulin-like peptide (ILP) localization in β-cells. Immuno-gold labeling for insulin using an antibody that recognizes all ILP on pancreatic sections from non-diabetic mice. Solid line boxes show double membrane organelles, likely autophagosomes, containing dispersed ILP. Dashed line box shows a lysososome engulfing a secretory granule and secretory granule core-like material. Size bar=500 nm. (B) Pancreatic sections of β-cell Atg7+/+ and Atg7−/− mice stained for proinsulin and P62/SQSTM1. Size bar=20 μm.

Autophagy regulates chromogranin-A in endocrine cells

We further studied whether autophagy also regulates other residents of the secretory pathway. CgA belongs to a family of large acidic secretory proteins found in most neuroendocrine tissues, including β-cells, stored with peptide hormones in secretory vesicles [25]. CgA is a large precursor peptide that, like proinsulin, undergoes proteolytic processing during its routing to and storage in secretory vesicles [26]. Inhibition of autophagy by Atg5/7-knockdown or treatment with bafilomycin-A1 markedly increased CgA expression in INS-1 β-cells, as well as in human pancreatic neuroendocrine cells (BON-1), whereas inhibition of proteasomal degradation had no effect (Figure 6). Increased CgA expression in BON-1 cells was accompanied by a small but significant increase of CgA secretion (Figure 6E), thus resembling proinsulin regulation by autophagy.

Figure 6.

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(A–D) Effects of inhibiting autophagic degradation by bafilomycin-A1, MG132 and Atg5/7-knockdown on CgA expression in INS-1E β-cells and BON-1 cells. Cells were treated with or without bafilomycin-A1 (A, C), MG132 (C) or transfected with scrambled siRNA or Atg5/7-siRNA (B, D) followed by cell extraction and measurement of CgA by Western blotting (n=6). (E) Effects of Atg5/7-knockdown on CgA secretion of BON-1 cells (n=3). * p<0.05.

Irreparable proinsulin mutants are refractory to autophagic degradation

The Akita proinsulin is trapped in the ER [23;27]. Akita proinsulin labeled with eGFP was transfected to INS-1E cells and its assignment for autophagic degradation analyzed by confocal microscopy and Western blotting (Figure 7). Intriguingly, Akita proinsulin was not found in autophagic punctae, including in cells in which autophagy was stimulated by rapamycin. Consistently, Akita proinsulin was not degraded in lysosomes, as evident by failure of bafilomycin-A1 to increase Akita proinsulin-eGFP expression (Figure 7B–C). The G(B23)V proinsulin mutant also generates ER stress; however, a small portion of this mutant may reach secretory granules [21]. In contrast to wild-type cells, bafilomycin-A1 failed to increase also G(B23)V-proinsulin expression; similarly, inhibition of the proteasome did not affect the steady-state proinsulin content (Figure 7C).

Figure 7.

Figure 7

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INS-1E cells were transfected with Akita proinsulin-eGFP (A–C) and G(B23)V proinsulin-eGFP constructs (C) followed by assessment of mutant proinsulin localization (A) and degradation (B–C). (A) Akita proinsulin-eGFP and P62/SQSTM1-mCherry colocalization following treatment with or without rapamycin or bafilomycin A1. Size bar =10 μm. (B) β-Cells expressing proinsulin-eGFP were treated with or without bafilomycin A1 at 3.3 and 22.2mmol/l glucose followed by Western blotting for LC3-II and for eGFP. Quantification of Akita proinsulin expression is shown below (n=3). (C) β-Cells expressing wild-type proinsulin, the Akita and G(B23)V proinsulin mutants were treated with and without bafilomycin-A1 or lactacystin. Quantification is shown below (n=3). * p<0.05.

Collectively, our findings show that irreparable proinsulin mutants are trapped in the ER, are not transported to autophagosomes and are refractory to lysosomal degradation.

Discussion

Our study suggests a previously unappreciated role for autophagy in β-cell proinsulin handling and insulin secretion. We show that a substantial amount of proinsulin is rapidly delivered to autophagosomes and directed to lysosomal degradation. Quantitatively, the impact of this pathway on proinsulin level is robust, since short-term inhibition of lysosomal degradation increased steady-state proinsulin content quite significantly. This was entirely unexpected, considering the background abundance of proinsulin in the β-cell. Indeed, while stimulation of β-cells with high glucose may induce up to 25-fold increase in proinsulin biosynthesis [3], steady-state proinsulin level remains nevertheless unchanged. Thus, the increase in proinsulin content following inhibition of lysosomal degradation shown here is indeed striking. Bafilomycin-A1 increased proinsulin by inhibiting its degradation; the changes in steady-state proinsulin level were not due to inhibition of its conversion to insulin, or to reduction of exocytosis. Bafilomycin-A1 may indeed affect vesicular pH, thereby impairing proinsulin processing and secretion; however, such alterations cannot account for the observed changes in proinsulin level: bafilomycin-A1 did not affect cumulative insulin secretion; moreover, inhibition of insulin secretion by diazoxide failed to increase proinsulin content. Increased proinsulin in bafilomycin-A1-treated cells and mouse islets was accompanied by a parallel increase in C-peptide, indicating that proinsulin was converted to insulin. Most importantly, genetic disruption of autophagy similarly increased proinsulin content, further suggesting that inhibition of lysosomal degradation is the main cause for the increase in β-cell proinsulin.

Several lines of evidence suggest that the canonical macroautophagy is the central degradation mechanism regulating proinsulin: EM revealed dispersed, non-granular ILP located in autophagosome-like structures, confocal microscopy showed proinsulin localization in LC3+ and P62/SQSTM1+ puncta and in protein aggregates that appears in autophagy deficient βAtg7 knockout mice. Moreover, knockdown of key ATGs increased, whereas genetic and pharmacologic stimulators of autophagy (tat-Beclin 1, rapamycin and trehalose) decreased proinsulin content.

CgA, a precursor of secretory hormones in different neuroendocrine cells, is also robustly degraded by autophagy, suggesting that autophagy is a key regulatory pathway of hormone precursors in endocrine cells. It has been previously shown that certain residents of the secretory pathway in β-cells, e.g. pancreatic prohormone convertases, are not degraded by autophagy [28], suggesting that the autophagic degradation of complex peptides like proinsulin and CgA is a selective process. The degradation of insulin and of proinsulin by lysosomes appears dissimilar. Insulin granule degradation is mainly mediated via microautophagy and crinophagy, whereas proinsulin degradation is mediated via macroautophagy. Pharmacological and genetic inhibition of autophagy increased proinsulin content without affecting insulin, hence the importance of lysosomal proinsulin degradation greatly outweighs that of insulin. It has been recently suggested that insulin granules may undergo autophagy-independent lysosomal degradation to keep insulin secretion low and avoid hypoglycemia in the fasting state [2]. Stimulation of autophagy by tat-Beclin1 increased insulin secretion under fasting conditions and in response to high glucose; however, the mechanisms involved are not clear. The authors did not study the effects of autophagy deficiency on insulin secretion and the possibility that tat-Beclin1 stimulated insulin secretion independent of autophagy was not excluded. We found that in β-cells autophagic flux is rapid, rather than being suppressed, both in KRBH and rich medium. Strikingly, increasing the proinsulin content by genetic disruption of autophagy was associated with a parallel increase of proinsulin and insulin secretion, indicating that autophagy restrains, rather than stimulates, secretion. Our findings are in agreement with Pearson et al, who also found that inhibiting autophagy enhanced insulin secretion (29). This may suggest that inhibition of proinsulin export to autophagosomes extends proinsulin residency in the secretory pathway followed by packaging into secretory granules, processing and secretion.

Intriguingly, the Akita proinsulin mutant, which is trapped in the ER and cannot be delivered to the Golgi and secretory granules [20;30;31], was resistant to lysosomal degradation; this may suggest that ER exit to the secretory pathway is essential for proinsulin degradation via autophagy to occur. Indeed, treatment with brefeldin-A, which prevents ER exit, abolished the lysosomal degradation of wild-type proinsulin. We found that proinsulin-containing autophagosomes expressed the TGN protein B4GALT1, hence we suggest that proinsulin delivery to autophagosomes takes place at the TGN. Further studies are required to uncover the precise location and the molecular mechanisms of selective proinsulin delivery to autophagosomes.

What is the biological rationale for proinsulin degradation by autophagy? In endocrine cells such as β-cells, intense hormone biosynthesis and flow through the ER-Golgi, along with highly dynamic changes in secretion, rationalizes the need for an effective post-ER degradation pathway that clears surplus hormone precursors in the resting state. It may be speculated that this may function as a “buffering” system aimed to prevent cargo overload at the Golgi, which may saturate the convertases with consequent prohormone over-secretion. Hence, autophagy can be viewed as a secretory pathway checkpoint that regulates hormone secretion.

The importance of autophagy for quality control is demonstrated by the fact that its disruption results in β-cell stress, cellular degeneration and impaired insulin secretion [10; 11], which may promote the progression from insulin resistance to diabetes [14]. Recently, it was shown that impaired autophagy led to accumulation of human islet-amyloid polypeptide (hIAPP) and exacerbated hIAPP-induced β-cell toxicity [32–34]. Hence, inhibiting autophagy might be a double-edged sword: increasing insulin secretion in the short-term, at the expense of ER stress and consequently β-cell degeneration and insulin deficiency in the long-term.

Overall, we show that autophagy is a post-ER pathway regulating proinsulin level and insulin secretion. The Golgi network may function as a bifurcation node determining proinsulin fate in the β-cell, assigning it either for further processing and secretion, or for degradation. Reduction of autophagy increases proinsulin retention in the secretory pathway, resulting in enhanced insulin secretion. The autophagic degradation of wild-type proinsulin has important pathophysiological implications for the development of insulin-deficiency in diabetes and highlights the potential for novel therapeutic strategies aimed to manipulate proinsulin clearance as a means to enhance insulin secretion in type 2 diabetes.

Supplementary Material

Supp Figures 1 - 9

Acknowledgments

This study was supported by grants from the Israel Science Foundation to GL (ISF-347/12), the Golda Meir foundation and the EFSD to JDW, and the NIH (NIH-DK48280) to PA.

We declare no potential conflicts of interest relevant to this article.

Y.R. researched data and edited the manuscript, J.D.W. researched data and edited the manuscript, E.B.-W. researched data, N.P. researched data, H.Z. researched data, M.-S. L., P.A., E.C. contributed to discussion and reviewed and edited the manuscript. W.Q., L.H., M.L. generated essential reagents for this study.

G.L. wrote the manuscript. G.L. is the guarantor of this work and, as such, had full access to all the data in the study and takes responsibility for the integrity of the data and the accuracy of the data analysis.

Abbreviations

ATG

autophagy gene

B4GALT1

B1,4-galactosyltransferase 1

CgA

chromogranin A

eGFP

enhanced green fluorescent protein

EM

electron microscopy

ER

endoplasmic reticulum

GAPDH

glyceraldehyde 3-phosphate dehydrogenase

hIAPP

human islet-amyloid polypeptide

HSP90

heat shock protein 90

ILP

insulin-like peptides

LAMP1

lysosomal-associated membrane protein 1

LC3

light chain microtubule-associated protein 3

mTORC1

mammalian target of rapamycin 1

PBA

4-phenylbutyric acid

SERCA

sarcoendoplasmic reticulum calcium transport ATPase

SQSTM1

sequestome 1

TGN

trans-Golgi network

UPR

unfolded protein response

WT

wild type

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Supplementary Materials

Supp Figures 1 - 9
NIHMS959110-supplement-Supp_Figures_1_-_9.pdf (1.4MB, pdf)

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