Lysosomal Ca2+-mediated TFEB activation modulates mitophagy and functional adaptation of pancreatic β-cells to metabolic stress

Park, Kihyoun; Lim, Hyejin; Kim, Jinyoung; Hwang, Yeseong; Lee, Yu Seol; Bae, Soo Han; Kim, Hyeongseok; Kim, Hail; Kang, Shin-Wook; Kim, Joo Young; Lee, Myung-Shik

doi:10.1038/s41467-022-28874-9

Download PDF

Article
Open access
Published: 14 March 2022

Lysosomal Ca²⁺-mediated TFEB activation modulates mitophagy and functional adaptation of pancreatic β-cells to metabolic stress

Nature Communications volume 13, Article number: 1300 (2022) Cite this article

9182 Accesses
24 Citations
11 Altmetric
Metrics details

Subjects

Abstract

Although autophagy is critical for pancreatic β-cell function, the role and mechanism of mitophagy in β-cells are unclear. We studied the role of lysosomal Ca²⁺ in TFEB activation by mitochondrial or metabolic stress and that of TFEB-mediated mitophagy in β-cell function. Mitochondrial or metabolic stress induced mitophagy through lysosomal Ca²⁺ release, increased cytosolic Ca²⁺ and TFEB activation. Lysosomal Ca²⁺ replenishment by ER- > lysosome Ca²⁺ refilling was essential for mitophagy. β-cell-specific Tfeb knockout (Tfeb^Δβ-cell) abrogated high-fat diet (HFD)-induced mitophagy, accompanied by increased ROS and reduced mitochondrial cytochrome c oxidase activity or O₂ consumption. Tfeb^Δβ-cell mice showed aggravation of HFD-induced glucose intolerance and impaired insulin release. Metabolic or mitochondrial stress induced TFEB-dependent expression of mitophagy receptors including Ndp52 and Optn, contributing to the increased mitophagy. These results suggest crucial roles of lysosomal Ca²⁺ release coupled with ER- > lysosome Ca²⁺ refilling and TFEB activation in mitophagy and maintenance of pancreatic β-cell function during metabolic stress.

Small extracellular vesicles from young plasma reverse age-related functional declines by improving mitochondrial energy metabolism

Article Open access 16 April 2024

Xiaorui Chen, Yang Luo, … Xi Chen

Integrated proteomics reveals autophagy landscape and an autophagy receptor controlling PKA-RI complex homeostasis in neurons

Article Open access 10 April 2024

Xiaoting Zhou, You-Kyung Lee, … Zhenyu Yue

A guide to cell death pathways

Article 18 December 2023

Junying Yuan & Dimitry Ofengeim

Introduction

Mitochondrial function is essential for insulin release and survival of pancreatic β-cells¹. Mitochondrial function is maintained by several factors such as mitochondrial biogenesis, fission, fusion, and selective autophagy (mitophagy). The importance of autophagy in the maintenance of β-cell function and mitochondria has been demonstrated in previous studies using β-cell-specific autophagy knockout (KO) (Atg7^Δβ-cell) mice which had reduced β-cell mass and mitochondrial abnormalities². However, the role and mechanism of mitophagy in pancreatic β-cells have not been clearly demonstrated.

Transcription factor EB (TFEB) acts as a master regulator of lysosome biogenesis and autophagy gene expression³. TFEB family members also play a role in mitophagy^4,5. In TFEB activation and mitophagy by mitochondrial stressors, lysosomal Ca²⁺ release plays an important part⁵. However, the mechanism that perpetuates lysosomal Ca²⁺ release and enables the full progression of mitophagy despite the small lysosomal volume⁶ is not clear.

We investigated the mechanism and functional role of mitophagy in pancreatic β-cells and found that TFEB is activated by mitochondrial reactive oxygen species (ROS)-induced lysosomal Ca²⁺ release through calcineurin. We also obtained data indicating the importance of ER → lysosome Ca²⁺ refilling in the sustained release of lysosomal Ca²⁺, TFEB activation and mitophagy by mitochondrial stressors. Finally, we observed that TFEB contributes to β-cell mitophagy through induction of mitophagy receptors such as Calcoco2 (Ndp52) and Optn.

Results

Mitophagy and TFEB nuclear translocation by mitochondrial stressors

We first studied whether mitophagy occurs in INS-1 insulinoma cells treated with mitochondrial stressors. Immunoblotting showed that INS-1 cells express Parkin (Supplementary Fig. 1a), which is necessary for mitophagy by mitochondrial stressors⁴. When we transfected cells with pMito-Keima encoding a pH-sensitive fluorescent probe with a mitochondria-targeting sequence⁷, tubular mitochondrial Keima fluorescence excited by 440-nm laser was observed. After rotenone treatment, a mitochondrial complex I inhibitor, many cells showed punctate fluorescence excited by a 590-nm laser (acidic puncta), indicating lysosomal delivery of mitochondrial Keima and mitophagy (Fig. 1a). When cells were treated with another mitochondrial stressor antimycin A, a mitochondrial complex III inhibitor, combined with oligomycin, an ATP synthase inhibitor (O/A)⁴, acidic Mito-Keima puncta were again well observed, indicating mitophagy (Fig. 1a). To confirm mitophagy, cells were transfected with mRFP-LC3 and treated with rotenone or O/A. In many cells, mRFP-LC3 puncta colocalized with TOM20, a mitochondrial outer membrane protein, were observed by immunofluorescence (Fig. 1b), supporting rotenone- or O/A-induced mitophagy in INS-1 cells.

**Fig. 1: Mitophagy after treating INS-1 cells with mitochondrial stressors.**

We next studied the role of TFEB, a master regulator of lysosomal biogenesis and autophagy genes³, in mitophagy because transcriptional control of autophagy/lysosomal genes could be important in mitophagy⁴. When we studied TFEB nuclear translocation, a crucial event in TFEB activation, using TFEB-GFP-transfected INS-1 cells, TFEB nuclear fluorescence was apparent after rotenone or O/A treatment (Fig. 1c), similar to previous results using HeLa cells⁴. When INS-1 cells transfected with GFP conjugated to TFE3, another member of the MiTF/TFE family (TFE3-GFP)⁸, TFE3 nuclear fluorescence was clearly observed after rotenone or O/A treatment as well (Fig. 1c). Nuclear translocation of endogenous TFEB and TFE3 was also observed after rotenone or O/A treatment (Fig. 1d). To study the role of TFEB/TFE3 activation in mitophagy, Tfeb- or Tfe3-KO INS-1 cells generated by CRISP3/Cas9 technology were employed⁹ (Supplementary Fig. 1b). In these cells, mitophagy by rotenone or O/A was significantly reduced (Fig. 1e), showing an important role of Tfeb or Tfe3 in mitophagy by mitochondrial stressors.

Lysosomal Ca²⁺ in mitophagy

To study the mechanism of TFEB nuclear translocation by mitochondrial stressors, we examined whether mitochondrial stressor-induced mitophagy involves calcineurin, one of the most important phosphatases regulating TFEB activation in starvation¹⁰. When INS-1 cells transfected with TFEB-GFP and HA-ΔCnA-H151Q, a dominant-negative mutant of calcineurin¹¹, were treated with rotenone or O/A, TFEB nuclear translocation by mitochondrial stressors was significantly reduced (Fig. 2a, b), indicating the role of calcineurin in TFEB activation by mitochondrial stressors. We also studied the role of calcineurin in mitophagy. In cells transfected with mRFP-LC3 and HA-ΔCnA-H151Q and then treated with rotenone or O/A, mitophagy identified as mRFP-LC3 puncta colocalized with TOM20, was also significantly reduced (Fig. 2c, d), showing the role of calcineurin in mitophagy by mitochondrial stressors.

**Fig. 2: Role of Ca²⁺ and calcineurin in mitophagy.**

We next studied whether lysosomal Ca²⁺ release is involved in TFEB activation by mitochondrial stressors¹⁰. When INS-1 cells transfected with GCaMP3-ML1 encoding a lysosome-specific Ca²⁺ probe detecting perilysosomal Ca²⁺¹² were treated with rotenone or O/A, lysosomal Ca²⁺ release was not visualized; however, perilysosomal Ca²⁺ release by Gly-Phe β-naphthylamide, (GPN), a cathepsin C substrate inducing osmotic lysis of lysosomes¹², was significantly reduced by rotenone or O/A pretreatment, suggesting preemptying or release of lysosomal Ca²⁺ by rotenone or O/A (Fig. 2e), similar to results using carbonyl cyanide m-chlorophenylhydrazone (CCCP), a mitochondrial uncoupler⁵. We next studied changes in lysosomal Ca²⁺ content ([Ca²⁺]_Lys) using Oregon Green 488 BAPTA-1 Dextran. [Ca²⁺]_Lys of INS-1 cells was significantly reduced by rotenone or O/A (Fig. 2f), indicating mitochondrial stressor-induced lysosomal Ca²⁺ release. Indirect estimation of [Ca²⁺]_Lys by calculating area under the curve (AUC) of cytoplasmic Ca²⁺ fluorescence ([Ca²⁺]_i) tracing using Fura-2 after GPN treatment¹³, also showed significantly reduced [Ca²⁺]_Lys by rotenone or O/A (Fig. 2g). However, lysosomal pH was not changed by rotenone treatment, while O/A treatment induced a small increase of lysosomal pH (Supplementary Fig. 2a), suggesting that [Ca²⁺]_Lys decrease by mitochondrial stressor is unrelated to lysosomal pH changes. We next investigated whether rotenone or O/A increases [Ca²⁺]_i due to lysosomal Ca²⁺ release. [Ca²⁺]_i determined by a ratiometric method using Fura-2 was significantly increased after rotenone or O/A treatment (Fig. 2h), suggesting that lysosomal Ca²⁺ release by rotenone or O/A leads to increased [Ca²⁺]_i, calcineurin activation and TFEB activation. To confirm the role of increased [Ca²⁺]_i in TFEB translocation and mitophagy, we used BAPTA-AM, a cell-permeable Ca²⁺ chelator¹⁴. BAPTA-AM pretreatment significantly reduced the proportion of cells with nuclear TFEB and the number of acidic Mito-Keima puncta by rotenone or O/A (Fig. 2i and Supplementary Fig. 3a), indicating TFEB nuclear translocation and mitophagy by cytoplasmic Ca²⁺ released from lysosome.

Since increased [Ca²⁺]_i by rotenone or O/A induced TFEB nuclear translocation and mitophagy through calcineurin, a phosphatase, we next studied changes in TFEB dephosphorylation by rotenone or O/A. After treatment with rotenone or O/A, electrophoretic mobility of total TFEB was markedly increased, suggesting TFEB dephosphorylation (Supplementary Fig. 4a). Consistently, phosphorylation at S142, one of the most important TFEB phosphorylation sites³, was markedly reduced by rotenone or O/A (Supplementary Fig. 4a). We also studied phosphorylation at S211 of TFEB, another important phosphorylation site, using immunoprecipitation assay based on TFEB pS211 binding to 14-3-3 protein¹⁵. In immunoblotting, band intensity of 14-3-3 binding motif of TFEB, thus pS211-TFEB level¹⁵, in TFEB immunoprecipitate was markedly reduced by rotenone or O/A treatment of TFEB-GFP-transfectants (Supplementary Fig. 4b). Similarly, band intensity of 14-3-3 binding motif of TFE3, thus pS321-TFE3 level¹⁶, in TFE3 immunoprecipitate was markedly reduced by rotenone or O/A treatment of TFE3-GFP-transfectants (Supplementary Fig. 4b). In addition, we reproducibly observed reduced expression of total TFEB or TFE3 after O/A treatment (Supplementary Fig. 4b), suggesting possible degradation of TFEB or TFE3, while the mechanism and significance of these results are unknown.

We next studied the role of TRPML1 channel, a lysosomal Ca²⁺ channel that can be crucial in autophagy¹⁷. ML-SI3, a TRPML1 channel antagonist¹⁸, significantly reduced [Ca²⁺]_i increase by rotenone or O/A (Supplementary Fig. 3b), suggesting Ca²⁺ release by mitochondrial stressors through TRPML1. ML-SI3 also significantly reduced mitophagy by rotenone or O/A (Supplementary Fig. 3a). To obtain genetic evidence, we conducted knockdown (KD) of Mcoln1 encoding TRPML1. RT-PCR confirmed markedly reduced Mcoln1 expression by siMcoln1 transfection (Supplementary Fig. 3c). In Mcoln1-KD cells, rotenone- or O/A-induced mitophagy detected as acidic Mito-Keima puncta was significantly reduced (Supplementary Fig. 3d), demonstrating a crucial role of TRPML1 in mitochondrial stressor-induced mitophagy.

We next studied the mechanism of lysosomal Ca²⁺ release through TRPML1 by mitochondrial stressors. We examined ROS accumulation because ROS affects diverse ion channels, and can be generated by mitochondrial stressors in the vicinity of lysosome¹⁹. We observed strong ROS accumulation stained with CM-H2DCFDA by rotenone or O/A (Supplementary Fig. 5a). Pretreatment with N-acetylcysteine (NAC), an antioxidant, markedly reduced ROS accumulation by rotenone or O/A (Supplementary Fig. 5a). Intriguingly, NAC significantly inhibited rotenone- or O/A-mediated increase of [Ca²⁺]_i (Supplementary Fig. 5b), which suggests that ROS induces lysosomal Ca²⁺ release and increases [Ca²⁺]_i, leading to TFEB nuclear translocation. We also studied changes in mitochondrial ROS that can be more relevant to the induction of mitophagy, using MitoSOX. Again, mitochondrial ROS was increased by rotenone or O/A, which was significantly reduced by MitoTEMPO, a mitochondrial ROS scavenger (Supplementary Fig. 5c). Furthermore, both the increase of [Ca²⁺]_i and the decrease of [Ca²⁺]_Lys by rotenone or O/A were suppressed by MitoTEMPO (Supplementary Fig. 5d, e), indicating the role of mitochondrial ROS in lysosomal Ca²⁺ efflux, increased [Ca²⁺]_i and decreased [Ca²⁺]_Lys by mitochondrial stressors.

Role of ER → lysosome Ca²⁺ refilling in mitophagy

Although [Ca²⁺]_Lys is comparable to [Ca²⁺]_ER, lysosome is a minor Ca²⁺ reservoir because of a small lysosomal volume and might not be a sufficient source to ensure full progression of intracellular events requiring Ca²⁺⁶. Therefore, we studied whether ER → lysosome Ca²⁺ movement that can follow lysosomal Ca²⁺ depletion²⁰, occurs during mitophagy. We observed that decreased [Ca²⁺]_Lys by rotenone or O/A recovered after mitochondrial stressor removal (Fig. 3a). Since ER, the largest Ca²⁺ reservoir in cells, is the most likely source replenishing depleted lysosomal Ca²⁺ store⁶, we conducted blockade of ER Ca²⁺ exit channels that could be routes for ER → lysosome Ca²⁺ movement. Xesto-spongin C, an inositol trisphosphate receptor (InsP3R) antagonist, significantly suppressed [Ca²⁺]_Lys recovery after mitochondrial stressor removal (Fig. 3a), suggesting the occurrence of ER → lysosome Ca²⁺ refilling during mitophagy through InsP3R. Dantrolene, an antagonist of ryanodine receptor, another ER Ca²⁺ exit channel, similarly inhibited [Ca²⁺]_Lys recovery after mitochondrial stressor removal (Fig. 3a), suggesting the involvement of both InsP3R and ryanodine receptor channels in ER → lysosome Ca²⁺ refilling in mitophagy. We next studied the effect of ER Ca²⁺ exit channel blockade on [Ca²⁺]_i increase by mitochondrial stressors. Both Xesto-spongin C and Dantrolene significantly attenuated [Ca²⁺]_i increases by mitochondrial stressors (Fig. 3b), suggesting that ER → lysosome Ca²⁺ refilling contributes to the increase of [Ca²⁺]_i by mitochondrial stressors. When we chelated ER Ca²⁺ with a membrane-permeant chelator N,N,N′,N′-tetrakis (2-pyridylmethyl)ethylene diamine (TPEN) that cannot chelate cytosolic Ca²⁺ due to low Ca²⁺ affinity²¹, both [Ca²⁺]_Lys recovery after mitochondrial stressor removal and [Ca²⁺]_i increase by mitochondrial stressors were reduced (Fig. 3a, b), supporting the role of ER Ca²⁺ in these processes. Furthermore, the number of Mito-Keima puncta after rotenone or O/A treatment was significantly reduced by Xesto-spongin C or Dantrolene (Fig. 3c), probably due to insufficient Ca²⁺ refilling. However, these results do not confirm the role of ER Ca²⁺ in these processes because Xesto-spongin C or Dantrolene might have off-target effects and TPEN can chelate Zn²⁺ in addition to ER Ca²⁺²². To further corroborate the role of ER Ca²⁺ in [Ca²⁺]_Lys recovery after mitochondrial stressor removal, we treated cells with Thapsigargin emptying ER Ca²⁺ store²⁰. After Thapsigargin pretreatment, [Ca²⁺]_Lys recovery after mitochondrial stressor removal was abrogated (Supplementary Fig. 6a), supporting the role of ER Ca²⁺ in [Ca²⁺]_Lys recovery and ER → lysosome Ca²⁺ refilling.

**Fig. 3: ER → lysosome Ca²⁺ refilling during mitophagy.**

Since these results suggest ER → lysosome Ca²⁺ movement during mitochondrial stress, we next measured ER Ca²⁺ content ([Ca²⁺]_ER) during mitophagy in INS-1 cells transfected with GEM-CEPIA1er²³. When [Ca²⁺]_ER was determined after rotenone or O/A treatment in a Ca²⁺-free Krebs-Ringer bicarbonate (KRB) buffer inhibiting possible store-operated Ca²⁺ entry (SOCE), a significant decrease of [Ca²⁺]_ER was observed (Supplementary Fig. 6b), consistent with ER → lysosome Ca²⁺ refilling. [Ca²⁺]_ER determined using a FRET-based D1ER probe allowing ratiometric measurement²⁴ also demonstrated a significantly reduced [Ca²⁺]_ER after rotenone or O/A treatment in a Ca²⁺-free KRB buffer (Fig. 3d). Importantly, [Ca²⁺]_ER was not changed by rotenone or O/A treatment in the presence of extracellular Ca²⁺ (Supplementary Fig. 6c). These results eliminate possible role of sarcoplasmic/endoplasmic reticulum Ca²⁺-ATPase (SERCA) inhibition due to rotenone- or O/A-induced decrease of ATP in [Ca²⁺]_ER decrease after mitochondrial stressor treatment without extracellular Ca²⁺ (see Fig. 3d), since [Ca²⁺]_ER would be decreased by SERCA inhibition even with extracellular Ca²⁺²³.

To study dynamic changes in [Ca²⁺]_ER and their temporal relationship with lysosomal Ca²⁺ refilling, we simultaneously traced [Ca²⁺]_ER and [Ca²⁺]_Lys in cells transfected with GEM-CEPIA1er and loaded with Oregon Green 488 BAPTA-1 Dextran. When cells were treated with rotenone or O/A and organelle [Ca²⁺] of cells with reduced [Ca²⁺]_Lys was monitored without rotenone or O/A in a Ca²⁺-free KRB buffer, [Ca²⁺]_ER decrease occurred simultaneously with [Ca²⁺]_Lys recovery (Fig. 3e), strongly supporting ER → lysosome Ca²⁺ refilling by mitochondrial stress. Without mitochondrial stressor removal, [Ca²⁺]_Lys recovery could not be observed probably due to ongoing lysosomal Ca²⁺ release (Supplementary Fig. 6d).

Since ER → lysosome Ca²⁺ refilling can be facilitated by membrane contact between organelles²⁵, we studied possible apposition of ER and lysosome proteins using proximity ligation assay (PLA). Confocal microscopy after incubation with specific antibodies (Abs) and rolling circle amplification showed close contact between VAPA on ER and ORP1L on lysosome²⁶ by rotenone or O/A (Fig. 3f), suggesting the formation of membrane contact sites between ER and lysosome by mitochondrial stressors, likely facilitating ER → lysosome Ca²⁺ refilling. We employed another method detecting ER-lysosome contact-a modification of Contact-ID technology using a split–pair system of pBir biotin ligase²⁷. When cells transfected with Flag-BirA(N-G78)-VAPA and STARD3-BirA(G79-C)-HA were treated with rotenone or O/A, biotin conjugate formation was observed after incubation with FITC-streptavidin, indicating contact between ER (VAPA) and lysosome (STARD3) (Supplementary Fig. 6e).

We next studied possible STIM1 oligomerization which can occur after ER Ca²⁺ emptying and lead to SOCE activation through Ca²⁺ release-activated channel (CRAC)²⁸. Indeed, STIM1 puncta were clearly observed after rotenone or O/A treatment (Supplementary Fig. 7a), supporting ER Ca²⁺ emptying by mitochondrial stressors. Furthermore, STIM1 puncta colocalized with Orai1, suggesting Ca²⁺ influx through SOCE²⁹ during mitophagy (Supplementary Fig. 7a). To confirm the role of extracellular Ca²⁺ influx through Orai1 in mitophagy, we employed CRAC inhibitors. When we treated cells with BTP2 inhibiting Orai1^30,31, mitophagy by rotenone or O/A was significantly reduced (Supplementary Fig. 7b). When we depleted extracellular Ca²⁺ content using 2 mM EGTA reducing [Ca²⁺]_i approximately to 161 nM (https://somapp.ucdmc.ucdavis.edu/pharmacology/bers/maxchelator/CaEGTA-TS.htm)³², mitophagy by rotenone or O/A was again significantly reduced (Supplementary Fig. 7b), supporting the importance of extracellular Ca²⁺ influx in mitophagy. [Ca²⁺]_Lys recovery after mitochondrial stressor removal was also reduced by incubation in a Ca²⁺-free KRB buffer or 2 mM EGTA (Supplementary Fig. 6a), suggesting the role of extracellular Ca²⁺ influx in ER → lysosome Ca²⁺ refilling.

Role of Tfeb and mitophagy in β-cell function

We next studied in vivo role of TFEB-mediated mitophagy in pancreatic β-cell function. To this end, we generated Tfeb^Δβ-cell mice by crossing Tfeb-floxed (Tfeb^F/F) mice to RIP-Cre mice, and imposed metabolic stress by feeding HFD. In this model, we wanted to confirm that metabolic stress can cause mitochondrial stress leading to mitophagy. We thus treated INS-1 cells with palmitic acid (PA), the most abundant saturated fatty acid in vivo and an effector of insulin resistance or β-cell failure^33,34. PA treatment of INS-1 cells induced mitochondrial stress, as shown by mitochondrial ROS accumulation (Fig. 4a). PA treatment of INS-1 cells also induced a decrease of [Ca²⁺]_Lys and an increase of [Ca²⁺]_i (Fig. 4b, c), suggesting that PA-induced mitochondrial ROS influences lysosomal Ca²⁺ channel. Lysosomal pH was not changed by PA treatment of INS-1 cells (Supplementary Fig. 2b), suggesting that PA-induced [Ca²⁺]_Lys decrease was not due to lysosomal pH change. Furthermore, TFEB nuclear translocation and mitophagy detected as acidic Mito-Keima puncta were observed after PA treatment (Fig. 4d, e), probably due to TFEB activation by increased [Ca²⁺]_i. PA-induced TFEB nuclear translocation was significantly attenuated by NAC or MitoTEMPO (Fig. 4f), suggesting the role of mitochondrial ROS in PA-induced TFEB activation. In contrast, autophagic activity represented as red puncta after mRFP-GFP-LC3 transfection was not increased by PA (Supplementary Fig. 2c), which is consistent with previous results^35,36 and suggests that changes of intracellular milieu and associated cellular responses after mitochondrial stressor treatment might affect flux of autophagy or selective autophagy differently. ER-phagic flux represented as red puncta after mCherry-GFP-RAMP4 transfection³⁷ was also not increased but decreased by PA (Supplementary Fig. 2d), consistent with a previous paper³⁸.

**Fig. 4: Effect of PA on mitochondria and mitophagy.**

While these results suggested the role of PA-induced mitochondrial ROS in mitophagy, PA can affect other signaling pathway such as fatty acid receptors belonging to G protein-coupled receptors (GPCRs) family³⁹ or can be converted to other lipid intermediates through palmitoyl-CoA or sphingosine/ceramide pathway⁴⁰. We thus examined the effect of inhibitors of such pathways. Pretreatment with DC260126 (a GPCR antagonist)³⁹, Triacsin C (an acyl-CoA synthetase inhibitor), Fumonisin B1 (a sphingosine N-acyltransferase inhibitor) or Myriocin (an inhibitor of serine palmitoyltransferase, thus ceramide synthesis)⁴⁰ did not reduce PA-induced mitochondrial ROS accumulation or TFEB nuclear translocation (Supplementary Fig. 8a, b), suggesting that mitochondrial ROS accumulation by PA itself is the most likely event leading to mitophagy.

Since effector molecules of metabolic stress such as PA can induce mitophagy through TFEB activation in vitro, we investigated whether mitophagy occurs in vivo after HFD feeding in pancreatic islet cells expressing Parkin (Supplementary Fig. 1c). Indeed, colocalization of LC3_, an autophagic marker, with TOM20 in pancreatic islets of Tfeb^F/F mice assessed by Pearson’s correlation analysis was markedly increased by HFD (Fig. 5a), indicating mitophagy. Intriguingly, increased mitophagy in islets of HFD-fed mice was significantly reduced by β-cell Tfeb KO (Fig. 5a), suggesting TFEB-dependent mitophagy by metabolic stress. As the number of LC3 autophagosomes might not reflect autophagic or mitophagic activity, we studied TOM20 colocalization with a lysosomal marker, as an indicator of mitophagic activity⁴¹. Mitophagic activity estimated by colocalization of TOM20 with LAMP2 was markedly increased in islets of HFD-fed Tfeb^F/F mice. Furthermore, the increased mitophagic activity in islets of HFD-fed mice was significantly reduced by β-cell Tfeb KO (Fig. 5b), suggesting TFEB-dependent increase in β-cell mitophagic activity by HFD. Electron microscopy (EM) also demonstrated decreased aspect ratio of mitochondria⁴² in β-cells of HFD-fed Tfeb^F/F mice, suggestive of mitophagy⁴³, which was reversed by β-cell Tfeb KO (Supplementary Fig. 9a). The number of mitochondria surrounded by isolation membrane or autophagosome in β-cells of HFD-fed Tfeb^F/F mice was also significantly increased compared to that of normal chow diet (NCD)-fed Tfeb^F/F or HFD-fed Tfeb^Δβ-cell mice (Supplementary Fig. 9b). In contrast, autophagic activity determined by colocalization between LAMP2 and LC3⁴⁴ was not significantly changed by HFD feeding or Tfeb KO (Supplementary Fig. 9c), which is in agreement with no increase of autophagic activity by PA in vitro and increased mitophagic activity without increased autophagic activity in cardiac tissue by HFD for 2 months⁴⁵. The number of autophagosome counted by EM was also not changed by HFD or Tfeb KO (Supplementary Fig. 9d).

**Fig. 5: Effect of in vivo *Tfeb* KO on mitophagy in β-cells.**

Probably due to decreased mitophagy, ROS accumulation detected by 4-hydroxynonenal (HNE) staining that was increased in islets of Tfeb^F/F mice on HFD was further increased by β-cell Tfeb KO (Fig. 5c). Regarding mitochondrial activity, cytochrome c oxidase (COX) activity in pancreatic islets was increased by HFD probably as an adaptation to metabolic stress, which was significantly reduced by β-cell Tfeb KO (Fig. 5d), suggesting TFEB-dependent functional mitochondrial adaptation to metabolic stress. Seahorse respirometry also showed significant increases of basal, glucose-stimulated, ATP-coupled and maximal O₂ consumption in pancreatic islets of HFD-fed mice again probably as an adaptation to metabolic stress. These increases of O₂ consumption were abolished by Tfeb KO (Fig. 5e), supporting TFEB-dependent adaptive response to metabolic stress.

Metabolically, NCD-fed Tfeb^Δβ-cell mice showed no abnormality in glucose profile up to 20 weeks of age (Supplementary Fig. 10a). However, HFD-fed Tfeb^Δβ-cell mice showed aggravated glucose intolerance compared to HFD-fed Tfeb^F/F mice, judged by increased AUC of glucose tolerance test (GTT) (Fig. 6a), while non-fasting blood glucose during or after HFD feeding for 20 weeks did not significantly differ between them (Supplementary Fig. 10a). Aggravated glucose intolerance of HFD-fed Tfeb^Δβ-cell mice was accompanied by reduced β-cell function compared to HFD-fed Tfeb^F/F mice as shown by decreased insulinogenic index (Fig. 6b) without significant changes of β-cell mass (Supplementary Fig. 10b), suggesting that Tfeb-KO β-cells could not functionally adapt to increased demand for insulin due to insufficient mitophagy and mitochondrial activity, leading to deteriorated glucose intolerance when metabolic stress was imposed. In contrast, AUC of insulin tolerance test (ITT) and body weight did not differ between Tfeb^Δβ-cell and Tfeb^F/F mice on either NCD or HFD (Supplementary Fig. 10c, d).

**Fig. 6: Effect of β-cell-specific *Tfeb* KO on metabolic profile and target gene expression.**

While these results demonstrated the role of TFEB in β-cell function and metabolic profile, TFE3 that is activated by mitochondrial stress might also play a non-redundant role in β-cell response to metabolic stress. We thus crossed Tfeb^Δβ-cell mice to Tfe3-KO mice. While Tfeb^F/F/Tfe3-KO and Tfeb^Δβ-cell/Tfe3-KO mice had slightly impaired glucose tolerance compared to Tfeb^F/F/Tfe3-WT mice and Tfeb^Δβ-cell/Tfe3-WT, respectively, these differences were statistically insignificant in both NCD- and HFD-fed conditions (Supplementary Fig. 10e), suggesting no further independent role of endogenous TFE3 in metabolic adaptation of β-cells.

Using Tfeb^Δβ-cell mice, we studied which genes are differentially regulated by Tfeb. Expression of Tfeb downstream genes related to autophagy such as LC3 (Map1lc3b), Clcn7 and Ctsa was upregulated in pancreatic islets of HFD-fed mice together with increased expression of Tfeb and Tfe3 themselves (Fig. 6c). Increased expression of these genes was significantly reduced by β-cell Tfeb KO (Fig. 6c), suggesting TFEB-dependent downstream gene induction in pancreatic islets by HFD. Intriguingly, expression of Ndp52 and Optn, crucial autophagy receptors related to mitophagy⁴⁶, was significantly increased in pancreatic islets of HFD-fed Tfeb^F/F mice, and these increases were markedly reduced by β-cell Tfeb KO (Fig. 6c), which could be related to TFEB-dependent increase of mitophagy by HFD. Expression of other potential mitophagy receptors, such as Nbr1, Tbk1 or Tax1bp1, in pancreatic islets was also increased by HFD, but these increases were statistically insignificant (Fig. 6c). Intriguingly, the expression of mitochondrial biogenesis genes such as Tfam, ESRRα or PGC-1α was increased in pancreatic islets of Tfeb^F/F mice by HFD. Such induction of mitochondrial biogenesis genes by HFD was abrogated by β-cell Tfeb KO (Fig. 6c), which is similar to previous results showing Tfeb-dependent induction of mitochondrial biogenesis genes in other tissues⁴⁷. Probably due to increased mitochondrial biogenesis in pancreatic islet cells by HFD, normalized density of TOM20 band in islets of Tfeb^F/F mice on HFD was not different from that of the same mice on NCD despite significantly increased mitophagy which can reduce mitochondrial proteins including TOM20⁴ (Supplementary Fig. 10f).

We next imposed mitochondrial stress on pancreatic islets ex vivo. In rotenone- or O/A-treated islets, mRNA expression of Ndp52, Optn and other autophagy or lysosomal genes such as Ctsa, Atp6v1h, Tfeb or Tfe3 was significantly increased (Fig. 6d). Furthermore, increases in Ndp52, Optn, Tfeb and Atp6v1h expression by ex vivo rotenone or O/A treatment were significantly reduced by β-cell Tfeb KO (Fig. 6d), supporting that TFEB-dependent induction of crucial mitophagy receptors and lysosomal genes by mitochondrial or metabolic stress facilitates mitophagy.

Since these results suggest that Ndp52 and Optn are potential TFEB targets, we conducted chromatin immunoprecipitation (ChIP) assay. To this end, we studied promoters of human NDP52 and OPTN that are well characterized⁴⁸. When 1.1B4 human islet cells generated by electrofusion of human primary pancreatic islet cells to pancreatic adenocarcinoma cells⁴⁹ were treated with rotenone or O/A, NDP52 or OPTN expression was significantly increased, which was downregulated by siTFEB (Fig. 6e, f). Contrarily, TFEB-GFP transfection significantly increased NDP52 or OPTN expression in 1.1B4 cells (Fig. 6g), supporting the validity of 1.1B4 cells to this end. In rotenone- or O/A-treated 1.1B4 cells, TFEB binding to putative Coordinated Lysosomal Expression and Regulation (CLEAR) sites⁵⁰ of NDP52 or OPTN promoter was significantly increased (Fig. 6h), supporting that NDP52 and OPTN are TFEB targets. Since Nfe2l2 encoding nuclear factor erythroid 2–related factor 2 (Nrf2) can be activated by TFEB or ROS, and Ndp52 and Optn could be Nfe2l2 target genes^48,51, we studied the role of Nfe2l2 in Ndp52 or Optn expression employing pancreatic islets from Nfe2l2-KO mice. Induction of Ndp52 or Optn in primary islet cells by rotenone or O/A was not significantly reduced by Nfe2l2 KO (Fig. 6i), suggesting that mitochondrial stressor-induced Ndp52 and Optn in pancreatic islet cells is largely due to TFEB-mediated transactivation. These results indicate that TFEB-dependent mitophagy, contributed by transactivation of mitophagy receptor genes such as Ndp52 or Optn, plays an important role in β-cell adaptation to metabolic stress through improved mitochondrial function.

Since the induction of Ndp52 and Optn by metabolic or mitochondrial stressors could explain selective mitophagy observed in pancreatic islets of HFD-fed mice, we conducted KD of Ndp52 and Optn in INS-1 cells that show increased expression of Ndp52 or Optn after treatment with PA (Supplementary Fig. 11a). Indeed, KD of Ndp52 and Optn that successfully reduced expression of Ndp52 and Optn, respectively (Supplementary Fig. 11b), significantly reduced mitophagy after PA treatment (Supplementary Fig. 11c), suggesting that induction of Ndp52 or Optn in β-cells contributes to the selective mitophagy after HFD feeding. The role of Ndp52 and Optn in mitophagy after rotenone or O/A treatment was also studied using INS-1 cells that show elevated expression of Ndp52 and Optn after rotenone or O/A treatment (Supplementary Fig. 11d). Mitophagy after rotenone or O/A treatment was also significantly reduced by KD of Ndp52 or Optn. Combined KD of both Ndp52 and Optn further reduced mitophagy after rotenone or O/A treatment (Supplementary Fig. 11e), suggesting that induction of Ndp52 or Optn contributes to mitophagy after treatment with mitochondrial stressors.

Discussion

Previous studies have shown the role of Tfeb family in systemic metabolism^47,52. However, the role of β-cell Tfeb in β-cell function and systemic metabolism has not been studied. Furthermore, while the role of autophagy in β-cell function and metabolism has been studied extensively, the role of mitophagy in β-cell function has been hardly investigated.

Mitophagy of pancreatic β-cells appears to be important in mitochondrial integrity and functional adaptation during metabolic or mitochondrial stress because HFD feeding increased insulinogenic index in wild-type mice but not in Tfeb^Δβ-cell mice that showed defective mitophagic response. Although the role of other types of selective autophagy or bulk autophagy modulated by TFEB cannot be disregarded in TFEB-mediated protection of β-cell function, flux of autophagy or ER-phagy was not increased by PA or HFD feeding. These results are consistent with previous papers showing no increase in fluxes of autophagy or ER-phagy in vitro and in vivo by PA or HFD feeding^36,38,45, which could be due to different intracellular environment and associated cellular responses imposed by diverse organelle stressors such as induction of mitophagy receptors, modifying TFEB-dependent or -independent autophagic activity.

TFEB-mediated mitophagy appears to be important in adaptive mitochondrial respiration because HFD-induced upregulation of mitophagy, COX activity, and mitochondrial O₂ consumption in β-cells was abolished by Tfeb KO. Increases in mitophagy, COX activity, and mitochondrial O₂ consumption in pancreatic islets of HFD-fed mice are in line with previous results reporting increased mitochondrial respiration, complex activities, or content and mitophagy in the heart or islet tissues after HFD feeding^45,53,54,55, which could be due to fatty acid-induced uncoupling, O₂ shunting to ROS or adaptive changes to metabolic stress⁵⁴. Probably due to impaired mitophagy and mitochondrial adaptation to metabolic stress, HFD-induced ROS accumulation was augmented by β-cell Tfeb KO, leading to significantly suppressed β-cell function and aggravated glucose intolerance. Consistent with HFD-induced increases of mitophagy and COX activity, induction of TFEB downstream genes or Tfeb and Tfe3 themselves was observed in pancreatic islets of HFD-fed mice, again supporting the role of TFEB activation in adaptation to HFD. However, we observed no further metabolic deterioration by additional Tfe3 KO despite comparable TFE3 activation by mitochondrial stressors, suggesting no non-redundant role of Tfe3 in β-cell adaptation to metabolic stress. These results are in contrast to a previous paper showing an independent role of Tfe3 in metabolic adaptation⁵⁶. Such discrepancies could be due to different types of tissues analyzed and non-identical experimental methods or mouse background.

Although these results suggest important roles of mitophagy in pancreatic β-cell function, previous papers have reported no significant decrease of glucose-stimulated insulin release from β-cells with targeted disruption of Parkin⁵⁷ or PINK1, an upstream kinase recruiting Parkin onto dysfunctional or damaged mitochondria^46,58. This discrepancy could be attributable to compensatory changes in Parkin- or PINK1-KO β-cells which might include Parkin-independent mitophagy such as piecemeal-type mitophagy or receptor-mediated mitophagy that can occur in Parkin-KO or PINK1-KO β-cells^59,60. In contrast, Parkin-KD INS-1 cells showed diminished mitochondrial function and glucose-stimulated insulin release⁶¹, which might be due to the absence of compensatory changes associated with Parkin KO. The role of β-cell Parkin in the maintenance of mitochondrial function and glucose-stimulated insulin release after treatment with streptozotocin, a classical β-cell toxin, has also been reported⁶², suggesting the role of Parkin-mediated mitophagy of β-cells in stressed condition.

We observed Ndp52 and Optn induction in pancreatic islets by metabolic or mitochondrial stress, which was attenuated by Tfeb KO. Since NDP52 and OPTN are primary receptors for mitophagy⁴⁶, TFEB-dependent induction of Ndp52 and Optn in pancreatic islet cells is likely to contribute to the increased mitophagy by metabolic stress or mitochondrial stress. These results suggest a role of transactivation of Ndp52 and Optn in mitochondrial stress-induced mitophagy besides mitophagy receptor phosphorylation⁶³.

When we studied the mechanism of mitophagy in β-cells undergoing mitochondrial stress, we observed significantly increased [Ca²⁺]_i by rotenone or O/A, similar to results using other cell types^64,65. As antimycin A had a relatively weak effect on mitochondrial potential, it has been used in combination with oligomycin inhibiting reverse activity of F₁F₀-ATPase⁶⁶. Ca²⁺ channel responsible for lysosomal Ca²⁺ release by mitochondrial ROS appears to be TRPML1. Besides PI(3,5)P₂, ROS can also activate TRPML1 channel but not TRPML2 or TRPML3 channel⁵. Since ML-SI3 is not specific for TRPML1, ML-SI3-mediated abrogation of mitochondrial stressor-induced [Ca²⁺]_i increase does not prove the role of TRPML1 in TFEB activation and mitophagy by mitochondrial stressors. However, lysosomal Ca²⁺ release by mitochondrial ROS that does not activate TRPML2 or TRPML3 channel and abrogation of mitochondrial stressor-induced TFEB activation by siMcoln1 strongly support the role of TRPML1 in mitochondrial stressor-induced lysosomal Ca²⁺ release. Following lysosomal Ca²⁺ release through TRPML1, increased [Ca²⁺]_i dephosphorylated TFEB and TFE3 via calcineurin activation. We also observed notably decreased total TFEB and TFE3 following O/A treatment. Although the mechanism of this phenomenon is unknown, it could be due to changes in the phosphorylation of amino acid residues related to the stability of TFEB family members⁶⁷.

Although the initial lysosomal Ca²⁺ release occurs in response to mitochondrial ROS, small lysosomal Ca²⁺ reservoir might not be sufficient to accomplish full progression of mitophagy⁶. While [Ca²⁺]_Lys is comparable to [Ca²⁺]_ER, lysosome is not a major Ca²⁺ reservoir because its volume is 1/10 of ER volume⁶. Previous papers demonstrated occurrence of ER → lysosome Ca²⁺ refilling for completion of events requiring lysosomal Ca²⁺²⁰. ER Ca²⁺ channels such as InsP3R or ryanodine receptor channel could act as ER Ca²⁺ exit channels in ER → lysosome Ca²⁺ refilling. Indeed, genetic studies have shown the participation of InsP3R in mitophagy⁶⁸; however, the role of ER Ca²⁺ or InsP3R in lysosomal Ca²⁺ refilling and the relationship between ER Ca²⁺ and lysosomal Ca²⁺ in mitophagy have not been studied. Our data show that ER → lysosome Ca²⁺ refilling occurs during mitochondrial stress, which is necessary for replenishment of lysosomal Ca²⁺ reservoir and completion of TFEB activation or mitophagy.

ER → lysosome Ca²⁺ refilling could be facilitated by ER-lysosome contact²⁵. In addition to well-characterized ER-mitochondrial contact, ER-lysosome contact could have a functional role in cellular responses⁶⁹. When ER Ca²⁺ emptying occurs due to ER → lysosome flux⁶, SOCE is likely to occur through CRAC activation, which is characterized by STIM1 oligomerization and translocation to ER-plasma junction where it interacts with Orai channels²⁸. Thus, STIM1 oligomerization by mitochondrial stressors strongly supports that ER Ca²⁺ emptying occurs due to ER → lysosome Ca²⁺ flux, which also can explain the suppression of mitophagy by extracellular Ca²⁺ depletion⁷⁰.

Altogether, these results suggest that TFEB is activated by lysosomal Ca²⁺ release coupled with ER → lysosome Ca²⁺ refilling during mitochondrial or metabolic stress, and TFEB-dependent induction of mitophagy, facilitated by transactivation of mitophagy receptors such as Ndp52 or Optn, plays an important role in β-cell adaptation to metabolic stress through improved mitochondrial function.

Methods

Detection of mitophagy in vitro

To visualize mitophagy using a mitophagy-specific probe, INS-1 cells were transfected with a pMito-Keima (MBL), and then treated with rotenone or O/A for 18 h. Keima fluorescence at neutral pH was observed at an excitation wavelength of 430 ± 20 nm and an emission wavelength of 624 ± 20 nm. Keima fluorescence in acidic conditions was observed at an excitation wavelength of 562 ± 20 nm and emission wavelength of 624 ± 20 nm. To visualize mitophagy in another way, cells transfected with the mRFP-LC3 construct were treated with mitochondrial stressors and then incubated with anti-TOM20 Ab (Cell Signaling Technology, 1:200). Images were obtained using an LSM780 confocal microscope (Zeiss) from more than five different fields for each experiment. Colocalization of puncta or fluorescence in vitro was evaluated manually in a blinded manner.

GCaMP3 Ca²⁺ imaging

INS-1 cells were transfected with a plasmid encoding a perilysosomal GCaMP3-ML1 Ca²⁺ probe¹². After 48 h, lysosomal Ca²⁺ release was measured in a zero Ca²⁺ solution (145 mM NaCl, 5 mM KCl, 3 mM MgCl₂, 10 mM glucose, 1 mM EGTA and 20 mM HEPES, pH 7.4) with or without rotenone or O/A pretreatment for 1 h by monitoring fluorescence intensity at 470 nm with a LSM780 confocal microscope (Zeiss). GPN was added at the indicated time.

Cytosolic Ca²⁺ measurement

For ratiometric measurement of [Ca²⁺]_i, cells were loaded with 2 μM of the acetoxymethyl ester form of Fura-2 (Invitrogen) in RPMI-1640 at 37 °C for 30 min and then rinsed with fresh solution. Measurements were performed using MetaFluor (Molecular devices) on an Axio Observer A1 (Zeiss) equipped with a 150 W xenon lamp Polychrome V (Till Photonics), a CoolSNAP-Hq2 digital camera (Photometrics), and a Fura-2 filter set. [Ca²⁺]_i was determined in a phenol red-free RPMI-1640 using the 340/380 nm fluorescence ratio and the following equation⁷¹.

$${[{{{{{{\rm{Ca}}}}}}}^{2+}]}_{{{{{{\rm{i}}}}}}}={K}_{d}\,{{{{{\rm{x}}}}}}\,[(R-{R}_{{{{{{\rm{min}}}}}}})/({R}_{{{{{{\rm{max}}}}}}}-R)]\,{{{{{\rm{x}}}}}}\,[{F}_{{{{{{\rm{min}}}}}}(380)}/{F}_{{{{{{\rm{max}}}}}}(380)}]$$

(1)

where K_d = Fura-2 dissociation constant (224 nM at 37 °C), F_min(380) = the 380 nm fluorescence in the absence of Ca²⁺, F_max(380) = 380 nm fluorescence with saturating Ca²⁺, R = 340/380 nm fluorescence ratio, R_max = 340/380 nm ratio with saturating Ca²⁺, and R_min = 340/ 380 nm ratio in the absence of Ca²⁺.

To monitor [Ca²⁺]_i after GPN treatment as an estimate of lysosomal Ca²⁺ content, cells were incubated in a zero Ca²⁺ solution containing 145 mM NaCl, 5 mM KCl, 3 mM MgCl₂, 10 mM glucose, 1 mM EGTA and 20 mM HEPES (pH 7.4)²⁰.

Measurement of lysosomal Ca²⁺ content ([Ca²⁺]_Lys)

To measure [Ca²⁺]_Lys, cells were loaded with 100 μg/mL of Oregon Green 488 BAPTA-1 Dextran (Invitrogen) at 37 °C for 12 h. Medium was then changed to one without Oregon Green 488 BAPTA-1 Dextran, and cells were chased for an additional 4 h. After treating cells with rotenone or O/A for the last 1 h of chasing, cells were washed in HEPES-buffered saline (135 mM NaCl, 5.9 mM KCl, 1.2 mM MgCl₂, 1.5 mM CaCl₂, 11.5 mM glucose, 11.6 mM HEPES, pH 7.3) for confocal microscopy²⁰.

Measurement of ER Ca²⁺ content ([Ca²⁺]_ER)

INS-1 cells were transfected with GEM-CEPIA1er (Addgene)²³ or a ratiometric FRET-based Cameleon probe D1ER²⁴ using Lipofectamine 2000. After 48 h, cells were treated with rotenone or O/A in a Ca²⁺-free KRB buffer (Sigma) for 1 h to inhibit influx of extracellular Ca²⁺ into ER³⁴ or in a culture medium for 1 h. GEM-CEPIA1er fluorescence was measured using an LSM780 confocal microscope (Zeiss) at an excitation wavelength of 405 nm and emission wavelengths of 466 or 520 nm, and F466/F520 was calculated as an index of [Ca²⁺]_ER²³. D1ER fluorescence intensity ratio (F540/F490) was determined using an LSM980 confocal microscope (Zeiss)

Simultaneous monitoring of [Ca²⁺]_Lys and [Ca²⁺]_ER

Twenty-four h after transfection with GEM-CEPIA1er, INS-1 cells were loaded with Oregon Green 488 BAPTA-1 Dextran for 12 h and chased for 4 h. After treating cells with rotenone or O/A for 1 h, cells were monitored for [Ca²⁺]_Lys and [Ca²⁺]_ER in a Ca²⁺-free KRB buffer in the presence or absence of rotenone or O/A using an LSM780 confocal microscope.

Lysosomal pH

After incubating cells with mitochondrial stressors or PA and 1 μM LysoSensor green DND-189 in a pre-warmed HBSS buffer (135 mM NaCl, 5.9 mM KCl, 1.2 mM MgCl₂, 1.5 mM CaCl₂, 11.5 mM glucose, 11.5 mM HEPES, pH 7.3) for 10 min in 37 °C incubator, LysoSensor green intensity was acquired using a confocal microscope and converted to pH using a standard curve obtained after treating cells with 10 μM nigericin for 20 min in 105 mM KCl buffer (105 mM KCl, 25 mM HEPES, 1 mM MgCl₂) adjusted to different pH⁷².

Proximity ligation assay (PLA)

Contact between ER and lysosome was studied using a Duolink InSitu Detection Reagents Red kit (Sigma) according to the manufacturer’s protocol. Briefly, cells were incubated with primary Abs against ORP1L (Abcam, 1:200) and VAPA (Santa Cruz, 1:200) at 4 °C overnight. After washing, cells were incubated with PLA plus and minus probes at 37 °C for 1 h. After ligation reaction to close the circle and rolling circle amplification (RCA) of the ligation product, fluorescence-labeled oligonucleotides hybridized to the RCA products were observed by fluorescence microscopy.

Contact-ID

Full-length DNA fragments of mouse VAPA and STARD3 were produced from cDNA from J774A.1 (ATCC, TIB-67) cells by PCR. DNA fragments encoding VAPA and STARD3 were inserted into Age1-XhoI site of Flag-BirA(N-G78) and Hind3-BamH1 site of BirA(G79-C)-HA vectors, respectively, using T4 DNA ligase (Takara). Nucleotide sequences of all constructs were confirmed by automatic DNA sequencing. Twenty-four h after transfection of INS-1 cells with Flag-BirA-VAPA and STARD3-BirA-HA using Lipofectamine 2000, 50 µM biotin was added for labeling overnight. After treatment with rotenone or O/A for 1 h, cells were fixed with 4% paraformaldehyde and then permeabilized with 0.5% Triton X-100-PBS. After 10% goat serum blocking, cells were sequentially incubated with anti-FLAG (Sigma, 1:200) and -HA Abs (Cell Signaling Technology, 1:200) diluted in PBS-1% BSA, secondary Abs diluted in PBS-1% BSA for 30 min, and then with FITC-streptavidin diluted in PBS-1% BSA for 30 min at room temperature. (VAPA-Age1 forward: CGACCGGTCATGGCGTCCGCCTCCGGG GCC ATG GC; VAPA-Xho1 reverse: GCCTCGAGCTACAAGATGAATTTCCCTAGAAAGAA; STARD3-Hind3 forward: CCAAGCTTATGAGCAAGCGACCTGGTGATCTGGCC; STARD3-BamH1 reverse: CGGATCCAGCTCGGGCCCCCAGCTCTCCAACACG).

CRAC activation

INS-1 cells transfected with YFP-STIM1 and 3 x FLAG-mCherry Red-Orai1/P3XFLAG7.1 (provided by Yuan J through Cha S-G) were treated with mitochondrial stressors for 30 min and then subjected to confocal microscopy to visualize STIM1 puncta and their colocalization with Orai1. Colocalization between STIM1 and Orai1 was confirmed by calculating Pearson’s correlation coefficient.

Cell culture and drug treatment

INS-1 cells (kindly provided by Wollheim C) were cultured in RPMI-1640 containing 11.2 mM glucose and 2 mM glutamine supplemented with 10% FBS, 1 mM pyruvate, 10 mM HEPES, 50 μM 2-mercaptoethanol, 100 U/mL penicillin and 100 μg/mL streptomycin. INS-1 cells were seeded onto wells of Poly-L-ornithine-coated plates. 1.1B4 cells obtained from ECACC (Salisbury) through Abdul Majid FA were cultured in RPMI-1640-10% FBS, 100 U/mL penicillin and 100 μg/mL streptomycin. For drug treatment, the following concentrations were used: 100 nM rotenone (Sigma), 200 nM oligomycin (Millipore), 125 nM antimycin A (Sigma), 400 μM PA (Sigma), 50 μM BAPTA-AM (Invitrogen), 20 μM ML-SI3, 200 μM GPN (Sigma), 3 μM Xesto-spongin C (Abcam), 10 μM Dantrolene (Sigma), 10 μM TPEN (Sigma), 10 μM BTP2 (Sigma), 2 mM EGTA (Sigma), 5 mM N-acetylcysteine (Sigma) and 100 μM MitoTEMPO (Sigma), 2 μM Thapsigargin (Sigma), 10 μM DC260126 (Cayman), 10 μM Fumonisin B1 (Sigma), 10 μM Myriocin (Sigma) and 5 μM Triacsin C (Abcam). PA stock solution (50 mM) was prepared by dissolving in 70% ethanol and heating at 55 °C⁴⁰. The working solution was made by diluting PA stock solution in 2% fatty acid-free BSA-RPMI 1640. To impose mitochondrial stress on primary pancreatic islets, higher doses of mitochondrial stressors (1 μM rotenone, 2 μM oligomycin, and 1.25 μM antimycin A) were employed.

Transfection and plasmids

Cells were transiently transfected with the following plasmids: MT-Keima, TFEB-GFP, TFE3-GFP, mRFP-LC3, mRFP-GFP-LC3, pcDNA3.1-HA, pcDNA3.1-HA-ΔCnA-H151Q, GCaMP3-ML1, GEM-CEPIA1er, YFP-STIM1, D1ER or mCherry-Orai1 using Lipofectamine 2000 (Invitrogen) according to the manufacturer’s protocol. In the case of TetOn-mCherry-GFP-RAMP4 (Addgene), cells were treated with 4 μg/ml Doxycycline for 48 h after transfection for 24 h before use. When HA-ΔCnA-H151Q was used, the same dose of regulatory calcineurin B subunit was transfected together. To evaluate autophagic and ER-phagic fluxes, the numbers of red puncta of transfected mRFP-GFP-LC3 and TetOn-mCherry-GFP-RAMP4³⁷ were counted, respectively.

Gene knockdown (KD)

INS-1 cells were transfected with Mcoln1 siRNA (CCCACAUCCAGGAGUGUAA), Ndp52 siRNA (CACACAGCAGAAAAUCCAA), Optn siRNA (CAGCUUACAGAUCUCAGUU) or control siRNA using Lipofectamine RNAiMAX (Invitrogen), according to the manufacturer’s protocol. 1.1B4 cells were transfected with TFEB siRNA (UGUAAUGCAUGACAGCCUG) or control siRNA using Lipofectamine RNAiMAX. Efficacy of Mcoln1 or TFEB siRNA KD was evaluated by RT-PCR. Primer sequences are listed in the Supplementary Tables 1, 3.

Abs and immunoblot analyses

Cells were solubilized in a lysis buffer containing protease inhibitors. Protein concentration was determined using the Bradford method. Samples (10–30 μg) were separated on 4–12% Bis-Tris gel (NUPAGE, Invitrogen) and transferred to nitrocellulose membranes for immunoblot analysis using the ECL method (Dongin LS). For immunoblotting, Abs against the following proteins were used: Parkin (Santa Cruz, 1:1,000), TFEB (Bethyl Laboratories, 1:1,000), TFE3 (Sigma Aldrich, 1:1,000), phospho-S142-TFEB (Millipore, 1:1,000), phospho-(Ser) 14-3-3 binding motif (Cell Signaling Technology, 1:1,000), HSP70 (Santa Cruz, 1:1,000), Tom20 (Cell Signaling Technology, 1:1,000), HSP90 (Abcam, 1:1,000) and ACTB (Santa Cruz, 1:1,000).

Immunoprecipitation

After lysis of Tfeb- or Tfe3-GFP-transfected INS-1 cells in an ice-cold lysis buffer (400 mM NaCl, 25 mM Tris-HCl, pH 7.4, 1 mM EDTA, and 1% Triton X-100) containing protease and phosphatase inhibitors, lysates were centrifuged at 12,000 g for 10 min in microfuge tubes, and supernatant was incubated with anti-GFP (AbFrontier, 1:1,000) in binding buffer (200 mM NaCl, 25 mM Tris-HCl, pH 7.4, 1 mM EDTA) with constant rotation at 4 °C for 1 h. After the addition of 50 μL of 50% of Protein-G bead (Roche Applied Science) slurry to lysates and incubation with rotation at 4 °C overnight, resins were washed with binding buffer. After resuspending pellet in a sample buffer (Life Technology) and heating at 100 °C for 3 min, supernatant was collected by centrifugation at 12,000 g for 30 sec for electrophoretic separation in a NuPAGE^® gradient gel (Life Technology). Immunoblotting was conducted by sequential incubation with anti-phospho-(Ser) 14-3-3 binding motif Ab (Cell Signaling Technology, 1:1,000) as the primary Ab and horseradish peroxidase-conjugated anti-rabbit IgG. Bands were visualized using an ECL kit for the detection of chemiluminescence.

Immunofluorescence

For TFEB and TFE3 immunofluorescence, cells treated with mitochondrial stressors were fixed with 4% paraformaldehyde and permeabilized with 0.5% Triton X-100. Cells were then blocked with 10% bovine serum albumin in PBS. After sequential incubation of cells with anti-TFEB (Bethyl Laboratories, 1:200) or anti-TFE3 Ab (Sigma, 1:200) at 4 °C overnight and then with anti-rabbit secondary Abs conjugated to Alexa Fluor 488 (Invitrogen) for 1 h, fluorescence was visualized with an LSM780 confocal microscope (Zeiss).

β-cell mass

Relative β-cell mass was determined by point counting morphometry after insulin immunohistochemistry using anti-insulin Ab (Cell Signaling Technology, 1:200)⁹. The number of test points was selected using a nomogram to set the probable error of volume density to less than 10% of the calculated value.

Electron Microscopy (EM)

After fixation in 2.5% glutaraldehyde-0.1 M NaH₂PO₄/Na₂HPO₄ phosphate buffer (pH 7.2) and phosphate buffer washing, pancreas tissues were post-fixed with 1% OsO₄ and embedded in Epon812. Ultrathin sections were mounted and stained with uranyl acetate/lead citrate (JEM-1011, Jeol). Aspect ratio was calculated as the ratio between the length of the major axis and that of the minor axis of mitochondria⁴². The number of mitochondria surrounded by isolation membrane or autophagosome was counted in 7~8 fields of magnified images (x 10,000) per mouse for a total of 25 fields as an index of mitophagy. The number of double-membrane structure with a luminal density similar to the cytoplasm but without identifiable organelle structure representing bulk autophagy was also counted in the same fields.

RNA extraction and real-time RT-PCR

Total RNA was extracted using TRIzol (Invitrogen), and cDNA was synthesized using M-MLV Reverse Transcriptase (Promega) according to the manufacturer’s protocol. Real-time RT-PCR was performed using SYBR green (Takara) in a QuantStudio3 Real-Time PCR System (Applied Biosystems). Expression values were normalized to the S18, GAPDH or Rpl32 mRNA level. Sequences of primers used for real-time RT-PCR are listed in Supplementary Tables 1, 2, and 3.

Measurement of intracellular ROS

To determine intracellular ROS, cells were treated with rotenone or O/A for 1 h after NAC pretreatment for 1 h. After incubation with 5 μM CM-H2DCFDA (Invitrogen) at 37 °C for 30 min in culture medium without FBS and recovery in complete medium for 10 min, confocal microscopy was conducted. To study mitochondria-specific ROS, cells were stained with 5 μM MitoSOX (Invitrogen) at 37 °C for 30 min. Cells were suspended in PBS-1% FBS, and then were subjected to flow cytometry by FACSVerse (BD Biosciences) using FlowJo software (TreeStar). The gating strategies for flow cytometric data analysis are shown in Supplementary Fig. 11.

ChIP assay

ChIP was performed according to the manufacturer’s protocol (SimpleChIP® Enzymatic Chromatin IP Kit, Cell Signaling). Briefly, the chromatin/DNA protein complexes were prepared by cross-linking chromatin of TFEB-GFP-transfected 1.1B4 cells treated with vehicle, rotenone, or O/A for 8 h by addition of 1% formaldehyde and incubation at room temperature for 10 min. Cell extracts were then sonicated to shear DNA and immunoprecipitated using anti-GFP (1:1,000) or IgG (1:50) with constant rotation at 4 °C overnight. After the addition of 30 μL of Protein-G bead slurry to the lysates and incubation with rotation at 4 °C for 2 h, resins were washed with the ChIP buffer supplied with the kit. After elution of chromatin from the Ab/Protein-G beads with ChIP Elution Buffer and reversing cross-linking by adding 5 M NaCl and 2 μL of 20 mg/mL proteinase K, DNA was purified for PCR using primers designed to cover the putative TFEB CLEAR sequence (CACGTG)⁵⁰ in the human NDP52 (-5,653 from the 5’ UTR) and OPTN promoters (−6885 from the 5’ UTR) (Supplementary Table 4). PCR consisted of 35-40 cycles of denaturation at 97 °C for 30 s, annealing at 65 °C and 55 °C for 30 s, and extension at 72 °C for 40 s for human NDP52 and OPTN promoter, respectively.

Animals

Tfeb^F/F mice were generated by breeding Tfeb^{tm1a(EUCOMM)Wtsi} mice (Mutant Mouse Resource and Research Center) with FLPeR mice (Wellcome Trust Sanger Institute). Tfeb^∆β-cell mice were generated by crossing Tfeb^F/F mice with RIP-Cre mice (Jackson Laboratory). Eight-week-old male Tfeb^∆β-cell mice were maintained in a 12-h light/12-h dark cycle and fed HFD for 12 weeks. During the observation period, mice were monitored for glucose profile and weighed. Tfe3-KO mice were from the Jackson Laboratory. All animal experiments were conducted in accordance with the Public Health Service Policy in the Humane Care and Use of Laboratory Animals. The mouse breeding temperature was 22 ± 2 °C, and the humidity was 50 ± 10%. Mouse experiments were approved by the IACUC of the Department of Laboratory Animal Resources of Yonsei University College of Medicine, an AAALAC-accredited unit.

Isolation of pancreatic islets

Primary murine islets were isolated from fasted mice using the collagenase digestion technique². Briefly, after injecting 2.5 ml of collagenase P (0.8 mg/ml) into the common bile duct, the pancreas was procured and incubated in a collagenase solution at 37 °C for 13 min 20 s. After cessation of enzymatic digestion with cold HBSS-5% FBS, tissue was passed through a 400 μm sieve and then centrifuged on 1.10, 1.085, 1.069, and 1.037 g/ml Biocoll gradients (Biochrom). Islets were collected from the interface using micropipettes.

Intraperitoneal GTT and insulinogenic index

Intraperitoneal GTT was performed by intraperitoneal injection of 1 g/kg of glucose after overnight fasting. Blood glucose concentrations were determined using a One Touch glucometer (Lifescan) before (0 min) and 15, 30, 60, 120, and 180 min after glucose injection. Insulinogenic index was calculated as follows: Δinsulin_15 min (pM)/Δglucose_15 min (mM). ITT was conducted by injecting intraperitoneally 1 U/kg of regular insulin to fasted mice and measuring blood glucose levels at 0, 15, 30, 60, and 120 min. Serum insulin was measured using Mouse insulin ELISA kit (Shibayagi).

Immunohistochemistry

Tissue samples were fixed in 10% buffered formalin and embedded in paraffin. Sections of 4 μm thickness were stained for immunohistochemical analyses using anti-LC3 (MBL, 1:200), -LAMP2 (Abcam, 1:200), -TOM20 (Cell Signaling Technology, 1:200), -insulin (Invitrogen, 1:200) or 4-HNE Abs (Abcam, 1:200), and then with anti-rabbit secondary Abs conjugated to Alexa Fluor 647 (Invitrogen), Alexa Fluor 594 (Invitrogen), Alexa Fluor 568 (Invitrogen), Alexa Fluor 405 (Invitrogen) or Alexa Fluor 488 (Invitrogen). COX staining was done by incubating unfixed cryosections of the pancreas in a reaction buffer containing 10 mg of cytochrome c, 5 mg of diaminobenzidine tetrahydrochloride (DAB), 9 ml of sodium phosphate buffer (0.1 M, pH 7.4) and 1 ml of catalase (20 μg/ml). COX activity was visualized with a standard DAB reaction. Images were acquired with a light microscope and analyzed using Fiji⁷³. To evaluate mitophagy in vivo, colocalization between LC3 and TOM20 or that between LAMP2 and TOM20 was assessed by calculating Pearson’s correlation coefficient. To evaluate autophagic flux in vivo, colocalization between LC3 and LAMP2 was determined in the same way.

Bioenergetic analysis of O₂ consumption

O₂ consumption of isolated mouse islets was measured using a Seahorse Extracellular Flux (XFe96) Analyzer (Agilent Technologies) equipped with a spheroid microplate-compatible thermal tray according to a modification of the manufacturer’s protocol. Briefly, islets were seeded into the wells of a poly-L-lysine-coated XF96 spheroid microplate (25 islets/well). Islet seeding was done by inserting a pipette tip directly into the wells of the spheroid microplate. Islets then were incubated with pre-warmed XF assay medium (Seahorse XF base DMEM supplemented with 3 mM glucose, 1% FBS, 1 mM sodium pyruvate and 2 mM glutamine) at 37 °C for 1 h in a non-CO₂ incubator. Basal respiration was measured in XF assay medium containing 3 mM glucose. Islets were then sequentially exposed to 20 mM glucose, 5 μM oligomycin, 1 μM FCCP and 5 μM rotenone/antimycin A at the indicated time points to measure glucose-stimulated, ATP-coupled, maximal and non-mitochondrial O₂ consumption, respectively. O₂ consumption was analyzed using the WaveTM software (Agilent Technologies).

Statistical analysis

All values are expressed as the means ± SEM of ≥ 3 independent experiments performed in triplicate. For densitometric analysis of immunoblot bands, the mean from 2 independent experiments was calculated. Two-tailed Student’s t test was used to compare values between two groups. One-way ANOVA with Tukey’s test was used to compare values between multiple groups. Two-way repeated-measures ANOVA with Bonferroni’s post-hoc test was used to compare multiple repeated measurements between groups. P values < 0.05 were considered to represent statistically significant differences.

Reporting summary

Further information on research design is available in the Nature Research Reporting Summary linked to this article.

Data availability

The data generated or analyzed during the current study and materials are available from the corresponding author without imposed restriction on reasonable request. Source data are provided with this paper.

References

Miki, T. et al. Defective insulin secretion and enhanced insulin action in KATP channel-deficient mice. Proc. Natl Acad. Sci. USA 95, 10402–10406 (1998).
Article ADS CAS PubMed PubMed Central Google Scholar
Jung, H. S. et al. Loss of autophagy diminishes pancreatic b-cell mass and function with resultant hyperglycemia. Cell Metab. 8, 318–324 (2008).
Article CAS PubMed Google Scholar
Settembre, C. et al. TFEB links autophagy to lysosomal biogenesis. Science 332, 1429–1433 (2011).
Article ADS CAS PubMed PubMed Central Google Scholar
Nezich, C. L., Wang, C., Fogel, A. & Youle, R. J. MiT/TFE transcription factors are activated during mitophagy downstream of Parkin and Atg5. J. Cell Biol. 210, 435–450 (2015).
Article CAS PubMed PubMed Central Google Scholar
Zhang, X. et al. MCOLN1 is a ROS sensor in lysosomes that regulates autophagy. Nat. Commun. 7, 12109 (2016).
Article ADS CAS PubMed PubMed Central Google Scholar
Penny, C. J., Kilpatrick, B. S., Han, J. M., Sneyd, J. & Patel, S. A computational model of lysosome-ER Ca²⁺ microdomains. J. Cell Sci. 127, 2934–2943 (2014).
CAS PubMed Google Scholar
Katayama, H., Kogure, T., Mizushima, N., Yoshimori, T. & Miyawaki, A. A sensitive and quantitative technique for detecting autophagic events based on lysosomal delivery. Chem. Biol. 18, 1042–1052 (2011).
Article CAS PubMed Google Scholar
Martina, J. A., Diab, H. I., Li, H. & Puertollano, R. Novel roles for the MiTF/TFE family of transcription factors in organelle biogenesis, nutrient sensing, and energy homeostasis. Cell. Mol. Life Sci. 71, 2483–2497 (2014).
Article CAS PubMed PubMed Central Google Scholar
Kim, J. et al. An autophagy enhancer ameliorates diabetes of human IAPP-transgenic mice through clearance of amyloidogenic oligomer. Nat. Commun. 12, 183 (2021).
Article CAS PubMed PubMed Central Google Scholar
Medina, D. L. et al. Lysosomal calcium signalling regulates autophagy through calcineurin and TFEB. Nat. Cell Biol. 17, 288–299 (2015).
Article PubMed PubMed Central Google Scholar
Cereghetti, G. M. et al. Dephosphorylation by calcineurin regulates translocation of Drp1 to mitochondria. Proc. Natl Acad. Sci. USA 105, 15803–15808 (2008).
Article ADS CAS PubMed PubMed Central Google Scholar
Shen, D. et al. Lipid storage disorders block lysosomal trafficking by inhibiting a TRP channel and lysosomal calcium release. Nat. Commun. 3, 731 (2012).
Article ADS PubMed Google Scholar
Coen, K. et al. Lysosomal calcium homeostasis defects, not proton pump defects, cause endo-lysosomal dysfunction in PSEN-deficient cells. J. Cell Biol. 198, 23–35 (2012).
Article CAS PubMed PubMed Central Google Scholar
Parekh, A. B. Ca²⁺ microdomains near plasma membrane Ca²⁺ channels: impact on cell function. J. Physiol. 586, 3043–3054 (2008).
Article CAS PubMed PubMed Central Google Scholar
Roczniak-Ferguson, A. et al. The transcription factor TFEB links mTORC1 signaling to transcriptional control of lysosome homeostasis. Sci. Signal 5, ra42 (2012).
Article PubMed PubMed Central Google Scholar
Martina, J. A. et al. The Nutrient-responsive transcription factor TFE3, promotes autophagy, lysosomal biogenesis, and clearance of cellular debris. Sci. Signal 7, 309 (2014).
Article Google Scholar
Wang, W. et al. Up-regulation of lysosomal TRPML1 channels is essential for lysosomal adaptation to nutrient starvation. Proc. Natl Acad. Sci. USA 112, E1373–E1381 (2015).
CAS PubMed PubMed Central Google Scholar
Samie, M. et al. A TRP channel in the lysosome regulates large particle phagocytosis via focal exocytosis. Dev. Cell 26, 511–524 (2013).
Article CAS PubMed PubMed Central Google Scholar
Bogeski, I. & Niemeyer, B. A. Redox regulation of ion channels. Antioxid. Redox Signal. 21, 859–862 (2014).
Article CAS PubMed PubMed Central Google Scholar
Garrity, A. G. et al. The endoplasmic reticulum, not the pH gradient, drives calcium refilling of lysosomes. eLife 5, e15887 (2016).
Article PubMed PubMed Central Google Scholar
Hofer, A. M., Fasolato, C. & Pozzan, T. Capacitative Ca²⁺ entry is closely linked to the filling state of internal Ca²⁺ stores: a study using simultaneous measurements of ICRAC and Intraluminal [Ca²⁺]. J. Cell Biol. 130, 325–334 (1998).
Article Google Scholar
Hawkins, B. E., Frederickson, C. J., Dewitt, D. S. & Prough, D. S. Fluorophilia: fluorophore-containing compounds adhere non-specifically to injured neurons. Brain Res. 1432, 28–35 (2012).
Article CAS PubMed Google Scholar
Suzuki, J., Kanemaru, K., Ishii, K., Ohkura, M. & Lino, M. Imaging intraorganellar Ca²⁺ at subcellular resolution using CEPIA. Nat. Commun. 5, 4153 (2014).
Article ADS CAS PubMed Google Scholar
Park, K. S., Poburko, D., Wollheim, C. B. & Demaurex, N. Amiloride derivatives induce apoptosis by depleting ER Ca²⁺ stores in vascular endothelial cells. Br. J. Pharma 156, 1296–1304 (2009).
Article CAS Google Scholar
Yang, J., Zhao, Z., Gu, M., Feng, X. & Xu, H. Release and uptake mechanisms of vesicular Ca²⁺ store. Protein Cell 10, 8–19 (2019).
Article CAS PubMed Google Scholar
Cianciola, N. L., Chung, S., Manor, D. & Carlin, C. R. Adenovirus modulates Toll-Like receptor 4 signaling by reprogramming ORP1L-VAP protein contacts for cholesterol transport from endosomes to the endoplasmic reticulum. J. Virol. 91, e01904-16 (2017).
Kwak, C. et al. Contact-ID, a tool for profiling organelle contact sites, reveals regulatory proteins of mitochondrial-associated membrane formation. Proc. Natl Acad. Sci. USA 117, 12109–12120 (2020).
Article CAS PubMed PubMed Central Google Scholar
Derler, I., Jardin, I. & Romanin, C. Molecular mechanisms of STIM/Orai communication. Am. J. Physiol. 310, C643–C662 (2016).
Article Google Scholar
Li, X. et al. Calmodulin dissociates the STIM1-Orai1 complex and STIM1 oligomers. Nat. Commun. 8, 1042 (2017).
Article ADS PubMed PubMed Central Google Scholar
Bogeski, I. et al. Pharmacology of ORAI channels as a tool to understand their physiological functions. Expert Rev. Clin. Pharmacol. 3, 291–303 (2010).
Article CAS PubMed Google Scholar
Zitt, C. et al. Potent inhibition of Ca²⁺ release-activated Ca²⁺ channels and T-lymphocyte activation by the pyrazole derivative BTP2. J. Biol. Chem. 279, 12427–12437 (2004).
Article CAS PubMed Google Scholar
Schoenmakers, T. J., Visser, G. J., Flik, G. & Theuvenet, A. P. CHELATOR: an improved method for computing metal ion concentrations in physiological solutions. Biotechniques 12, 870–874 (1992).
CAS PubMed Google Scholar
Coll, T. et al. Oleate reverses palmitate-induced insulin resistance and inflammation in skeletal muscle cells. J. Biol. Chem. 283, 11107–11116 (2008).
Article CAS PubMed Google Scholar
Xu, S. et al. Palmitate induces ER calcium depletion and apoptosis in mouse podocytes subsequent to mitochondrial oxidative stress. Cell Death Dis. 6, e1976 (2015).
Article CAS PubMed PubMed Central Google Scholar
Hernández-Cáceres, M. P. et al. Palmitic acid reduces the autophagic flux in hypothalamic neurons by impairing autophagosome-lysosome fusion and endolysosomal dynamics. Mol. Cell. Oncol. 25, 1789418 (2020).
Article Google Scholar
Koga, H., Kaushik, S. & Cuervo, A. M. Altered lipid content inhibits autophagic vesicular fusion. FASEB J. 24, 3052–3065 (2010).
Article CAS PubMed PubMed Central Google Scholar
Liang, J. R., Lingeman, E., Ahmed, S. & Corn, J. E. Atlastins remodel the endoplasmic reticulum for selective autophagy. J. Cell Biol. 217, 3354–3367 (2018).
Article CAS PubMed PubMed Central Google Scholar
Lim, Y., Kim, S. & Kim, E. K. Palmitate reduces starvation-induced ER stress by inhibiting ER-phagy in hypothalamic cells. Mol. Brain 14, 65 (2021).
Article CAS PubMed PubMed Central Google Scholar
Grundmann, M., Bender, E., Schamberger, J. & Eitner, F. Pharmacology of Free Fatty Acid Receptors and Their Allosteric Modulators. Int. J. Mol. Sci. 22, 1763 (2021).
Article CAS PubMed PubMed Central Google Scholar
Han, M. S. et al. Lysophosphatidylcholine as a death effector in lipoapoptosis of hepatocytes. J. Lipid Res. 49, 84–97 (2008).
Article CAS PubMed Google Scholar
Fang, E. F. et al. Mitophagy inhibits amyloid-β and tau pathology and reverses cognitive deficits in models of Alzheimer’s disease. Nat. Neurosci. 22, 401–412 (2019).
Article CAS PubMed PubMed Central Google Scholar
Picard, M., White, K. & Turnbull, D. M. Mitochondrial morphology, topology, and membrane interactions in skeletal muscle: a quantitative three-dimensional electron microscopy study. J. Appl. Physiol. 114, 161–171 (2013).
Article PubMed Google Scholar
Bhansali, S., Bhansali, A., Walia, R., Saikia, U. N. & Dhawan, V. Alterations in Mitochondrial Oxidative Stress and Mitophagy in Subjects with Prediabetes and Type 2 Diabetes Mellitus. Front. Endocrinol. 8, 347 (2017).
Article Google Scholar
Pi, H. et al. Enhancing lysosomal biogenesis and autophagic flux by activating the transcription factor EB protects against cadmium-induced neurotoxicity. Sci. Rep. 7, 43466 (2017).
Article ADS PubMed PubMed Central Google Scholar
Tong, M. et al. Mitophagy is essential for maintaining cardiac function during high fat diet-induced diabetic cardiomyopathy. Circ. Res. 125, 1360–1371 (2019).
Article Google Scholar
Lazarou, M. et al. The ubiquitin kinase PINK1 recruits autophagy receptors to induce mitophagy. Nature 524, 309–314 (2015).
Mansueto, G. et al. Transcription Factor EB controls metabolic flexibility during exercise. Cell Metab. 25, 182–196 (2015).
Article Google Scholar
Jo, C. et al. Nrf2 reduces levels of phosphorylated tau protein by inducing autophagy adaptor protein NDP52. Nat. Commun. 5, 3496 (2014).
Article ADS PubMed Google Scholar
McCluskey, J. T. et al. Development and functional characterization of insulin-releasing human pancreatic beta cell lines produced by electrofusion. J. Biol. Chem. 286, 21982–21992 (2011).
Article CAS PubMed PubMed Central Google Scholar
Doronzo, G. et al. TFEB controls vascular development by regulating the proliferation of endothelial cells. EMBO J. 38, e98250 (2019).
Article PubMed Google Scholar
Park, J. Y., Kim, S., Sohn, H. Y., Koh, Y. H. & Jo, C. TFEB activates Nrf2 by repressing its E3 ubiquitin ligase DCAF11 and promoting phosphorylation of p62. Sci. Rep. 9, 14354 (2019).
Article ADS PubMed PubMed Central Google Scholar
Settembre, C. et al. TFEB controls cellular lipid metabolism through a starvation-induced autoregulatory loop. Nat. Cell Biol. 15, 647–658 (2013).
Article CAS PubMed PubMed Central Google Scholar
Roat, R. et al. Alterations of pancreatic islet structure, metabolism and gene expression in diet-induced obese C57BL/6J mice. PLoS ONE 9, e86815 (2014).
Article ADS PubMed PubMed Central Google Scholar
Rocha, C., Koury, O. H., Scheede-Bergdahl, C. & Bergdahl, A. Cardiac mitochondrial respiration following a low-carbohydrate, high-fat diet in apolipoprotein E-deficient mice. J. Physiol. Biochem. 75, 65–72 (2019).
Article CAS PubMed Google Scholar
Yu, H.-T. et al. Oxidative damage of mitochondrial respiratory chain in different organs of a rat model of diet-induce obesity. Eur. J. Nutr. 57, 1957–1967 (2018).
Article CAS PubMed Google Scholar
Pastore, N. et al. TFEB and TFE3 cooperate in the regulation of the innate immune response in activated macrophages. Autophagy 12, 1240–1258 (2016).
Article CAS PubMed PubMed Central Google Scholar
Corsa, C. A. S. et al. The E3 ubiquitin ligase Parkin is dispensable for metabolic homeostasis in murine pancreatic β cells and adipocytes. J. Biol. Chem. 294, 7296–7307 (2019).
Article CAS PubMed PubMed Central Google Scholar
Deas, E. et al. PINK1 deficiency in β-cells increases basal insulin secretion and improves glucose tolerance in mice. Open Biol. 4, 144051 (2014).
Article Google Scholar
Le Guerroué, F. et al. Autophagosomal content profiling reveals an LC3C-dependent piecemeal mitophagy pathway. Mol. Cell 68, 786–796 (2017).
Article PubMed Google Scholar
Liu, L. et al. Mitochondrial outer-membrane protein FUNDC1 mediates hypoxia-induced mitophagy in mammalian cells. Nat. Cell Biol. 14, 177–185 (2012).
Article PubMed Google Scholar
Jin, H.-S. et al. The PARK2 gene is involved in the maintenance of pancreatic β-cell functions related to insulin production and secretion. Mol. Cell. Endocrinol. 382, 178–189 (2014).
Article CAS PubMed Google Scholar
Hoshino, A. et al. Inhibition of p53 preserves Parkin-mediated mitophagy and pancreatic β-cell function in diabetes. Proc. Natl Acad. Sci. USA 111, 3116–3121 (2014).
Article ADS CAS PubMed PubMed Central Google Scholar
Moore, A. S. & Holzbaur, E. L. F. Dynamic recruitment and activation of ALS-associated TBK1 with its target optineurin are required for efficient mitophagy. Proc. Natl Acad. Sci. USA 113, E3349–E3358 (2016).
Article CAS PubMed PubMed Central Google Scholar
Johnson, P. R., Tepikin, A. V. & Erdemli, G. Role of mitochondria in Ca²⁺ homeostasis of mouse pancreatic acinar cells. Cell Calcium 32, 59–69 (2002).
Article CAS PubMed Google Scholar
Liu, C. et al. Crosstalk between Ca²⁺ signaling and mitochondrial H₂O₂ is required for rotenone inhibition of mTOR signaling pathway leading to neuronal apoptosis. Oncotarget 7, 7534–7549 (2016).
Article PubMed PubMed Central Google Scholar
Georgakopoulos, N. D., Wells, G. & Campanella, M. The pharmacological regulation of cellular mitophagy. Nat. Chem. Biol. 13, 136–146 (2017).
Article CAS PubMed Google Scholar
Ferron, M. et al. A RANKL-PKCβ-TFEB signaling cascade is necessary for lysosomal biogenesis in osteoclasts. Genes Dev. 27, 955–969 (2013).
Article CAS PubMed PubMed Central Google Scholar
MacVicar, T. D., Mannack, L. V., Lees, R. M. & Lane, J. D. Targeted siRNA screens identify ER-to-mitochondrial calcium exchange in autophagy and mitophagy responses in RPE1 cells. Int. J. Mol. Sci. 16, 13356–13380 (2015).
Article CAS PubMed PubMed Central Google Scholar
Kilpatrick, B. S., Eden, E. R., Schapira, A. H., Futter, C. E. & Patel, S. Direct mobilisation of lysosomal Ca²⁺ triggers complex Ca²⁺ signals. J. Cell Sci. 126, 60–66 (2013).
Article CAS PubMed Google Scholar
Gómez-Sánchez, R. et al. Mitochondrial impairment increases FL-PINK1 levels by calcium-dependent gene expression. Neurobiol. Dis. 62, 424–440 (2015).
Google Scholar
Grynkiewicz, G., Poenie, M. & Tsien, R. Y. A new generation of Ca²⁺ indicators with greatly improved fluorescence properties. J. Biol. Chem. 260, 3440–3450 (1985).
Article CAS PubMed Google Scholar
Ghosh, S. et al. β-Coronaviruses use lysosomes for egress instead of the biosynthetic secretory pathway. Cell 183, 1520–1535 (2020).
Article CAS PubMed PubMed Central Google Scholar
Kim, K. H. et al. Autophagy deficiency leads to protection from obesity and insulin resistance by inducing Fgf21 as a mitokine. Nat. Med. 19, 83–92 (2013).
Article CAS PubMed Google Scholar

Download references

Acknowledgements

The authors thank Cha S-G and Park K-S for provision of probes for organelle Ca²⁺ measurement, and Jae-Ho Cheong for Seahorse respirometry. ΔCnA-H151Q mutant and mRFP-LC3 were gifts from Scorrano L and Yoshimori T, respectively. GCaMP3-ML1 plasmid and ML-SI3 were provided by Xu H. INS-1 cells were from Wollheim C. Nfe2l2-KO mice were obtained from Yamamoto M. Flag-BirA(N-G78) and BirA(G79-C)-HA backbone vectors were from Rhee H-W. This study was supported by the National Research Foundation of Korea (NRF) grant funded by the Korea government (MSIT) (NRF-2019R1A2C3002924) and by the Bio&Medical Technology Development Program (2017M3A9G7073521). M.-S.L. is the recipient of a grant from the Faculty Research Assistance Program of Yonsei University College of Medicine (6-2016-0055) and A3 Foresight Program of the NRF (2015K2A2A6002060).

Author information

Authors and Affiliations

Severance Biomedical Science Institute, Yonsei University College of Medicine, Seoul, Korea
Kihyoun Park, Hyejin Lim, Jinyoung Kim, Yeseong Hwang, Yu Seol Lee, Soo Han Bae & Myung-Shik Lee
Department of Health Sciences and Technology, SAIHST, Sungkyunkwan University School of Medicine, Seoul, Korea
Kihyoun Park
Department of Biochemistry, Chungnam National University, Daejeon, Korea
Hyeongseok Kim
Graduate School of Medical Science and Engineering, Korea Advanced Institute of Science and Technology, Daejeon, Korea
Hail Kim
Department of Internal Medicine, Yonsei University College of Medicine, Seoul, Korea
Shin-Wook Kang & Myung-Shik Lee
Department of Pharmacology and Brain Korea 21 Project for Medical Sciences, Seoul, Korea
Joo Young Kim
Soonchunhyang Institute of Medi-bio Science, Soonchunhyang University, Cheonan, Korea
Myung-Shik Lee

Authors

Kihyoun Park
View author publications
You can also search for this author in PubMed Google Scholar
Hyejin Lim
View author publications
You can also search for this author in PubMed Google Scholar
Jinyoung Kim
View author publications
You can also search for this author in PubMed Google Scholar
Yeseong Hwang
View author publications
You can also search for this author in PubMed Google Scholar
Yu Seol Lee
View author publications
You can also search for this author in PubMed Google Scholar
Soo Han Bae
View author publications
You can also search for this author in PubMed Google Scholar
Hyeongseok Kim
View author publications
You can also search for this author in PubMed Google Scholar
Hail Kim
View author publications
You can also search for this author in PubMed Google Scholar
Shin-Wook Kang
View author publications
You can also search for this author in PubMed Google Scholar
Joo Young Kim
View author publications
You can also search for this author in PubMed Google Scholar
Myung-Shik Lee
View author publications
You can also search for this author in PubMed Google Scholar

Contributions

M.-S.L. conceived the experiments. K.P., H.L., J.K., Y.H., Y.S.L., S.H.B., H. K., S.-W.K., and J.Y.K. conducted experiments. M.-S.L. and K.P. wrote the manuscript.

Corresponding author

Correspondence to Myung-Shik Lee.

Ethics declarations

Competing interests

M.-S.L. is the CEO of LysoTech, Inc. All authors declare no competing interests.

Peer review

Peer review information

Nature Communications thanks Hirotaka Watada and the other anonymous reviewer(s) for their contribution to the peer review this work. Peer reviewer reports are available.

Additional information

Publisher’s note Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.

Supplementary information

Supplementary Information

Peer Review File

Description of Additional Supplementary Information

Reporting Summary

Source data

Rights and permissions

Open Access This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The images or other third party material in this article are included in the article’s Creative Commons license, unless indicated otherwise in a credit line to the material. If material is not included in the article’s Creative Commons license and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this license, visit http://creativecommons.org/licenses/by/4.0/.

Reprints and permissions

About this article

Cite this article

Park, K., Lim, H., Kim, J. et al. Lysosomal Ca²⁺-mediated TFEB activation modulates mitophagy and functional adaptation of pancreatic β-cells to metabolic stress. Nat Commun 13, 1300 (2022). https://doi.org/10.1038/s41467-022-28874-9

Download citation

Received: 02 April 2021
Accepted: 28 January 2022
Published: 14 March 2022
DOI: https://doi.org/10.1038/s41467-022-28874-9

This article is cited by

TFEB activation triggers pexophagy for functional adaptation during oxidative stress under calcium deficient-conditions
- Laxman Manandhar
- Raghbendra Kumar Dutta
- Raekil Park
Cell Communication and Signaling (2024)
Regulation of autophagy by perilysosomal calcium: a new player in β-cell lipotoxicity
- Ha Thu Nguyen
- Andreas Wiederkehr
- Kyu-Sang Park
Experimental & Molecular Medicine (2024)
Lysosomal Ca2+ as a mediator of palmitate-induced lipotoxicity
- Soo-Jin Oh
- Yeseong Hwang
- Myung-Shik Lee
Cell Death Discovery (2023)
Pancreatic β-cell mitophagy as an adaptive response to metabolic stress and the underlying mechanism that involves lysosomal Ca2+ release
- Soo-Jin Oh
- Kihyoun Park
- Myung-Shik Lee
Experimental & Molecular Medicine (2023)
Reversing pancreatic β-cell dedifferentiation in the treatment of type 2 diabetes
- Jinsook Son
- Domenico Accili
Experimental & Molecular Medicine (2023)

Comments

By submitting a comment you agree to abide by our Terms and Community Guidelines. If you find something abusive or that does not comply with our terms or guidelines please flag it as inappropriate.