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PDK4 augments ER-mitochondria contact to dampen skeletal muscle insulin signaling during obesity

Themis Thoudam1,2*, Chae-Myeong Ha1,2*, Jaechan Leem3*, Dipanjan Chanda4*, Jong-Seok

Park5, Hyo-Jeong Kim6, Jae-Han Jeon4,7, Yeon-Kyung Choi4,7, Suthat Liangpunsakul 8,9,10,

Yang Hoon Huh6, Tae-Hwan Kwon1,2, Keun-Gyu Park4,7, Robert A. Harris9, Kyu-Sang

Park11, Hyun-Woo Rhee12 & In-Kyu Lee1,2,4,7

Affiliations

1Department of Biomedical Science, Graduate School, 2BK21 Plus KNU Biomedical

Convergence Program, Kyungpook National University, Daegu, Republic of Korea,

Department of Immunology, School of Medicine, 3Catholic University of Daegu, Daegu,

Republic of Korea, 4Leading-edge Research Center for drug Discovery & Development for

Diabetes & Metabolic Disease, Daegu, Republic of Korea, 5Department of Chemistry, Ulsan

National Institute of Science and Technology, Ulsan, Republic of Korea, 6Electron

Microscopy Research Center, Korea Basic Science Institute, Ochang, Chungbuk, Republic of

Korea, 7Department of Internal Medicine, School of Medicine, Kyungpook National

University, Daegu, Republic of Korea, 8Division of Gastroenterology and Hepatology,

Department of Medicine, Indiana University School of Medicine, Indianapolis, IN, USA,

9Department of Biochemistry and Molecular Biology, Indiana University School of Medicine,

Indianapolis, IN, USA, 10Roudebush Veterans Administration Medical Center, Indianapolis,

IN, USA, 11Department of Physiology, Institute of Lifestyle Medicine, Yonsei University

Wonju College of Medicine, Gangwon-Do, Republic of Korea, 12Department of Chemistry,

Seoul National University, Seoul, Republic of Korea.

*These authors contributed equally to this work.

1

Diabetes Publish Ahead of Print, published online December 6, 2018 Diabetes Page 2 of 91

Correspondence to

In Kyu Lee, M.D., Ph.D.

Division of Endocrinology and Metabolism, Department of Internal Medicine, Kyungpook

National University School of Medicine, 130 Dongdeok-ro, Jung-gu, Daegu, 41404, Republic of Korea. Tel.: +82-53-420-5564; Fax: +82-53-420-2046; E-mail: [email protected]

Word count: 5404

Number of Figures: 7

Short running title: PDK4-induced MAM formation promotes muscle IR

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ABSTRACT

Mitochondria-associated ER membrane (MAM) is a structural link between mitochondria

and endoplasmic reticulum (ER). MAM regulates Ca2+ transport from the ER to mitochondria

via an IP3R1-GRP75-VDAC1 complex-dependent mechanism. Excessive MAM formation

may cause mitochondrial Ca2+ overload and mitochondrial dysfunction. However, the exact

implication of MAM formation in metabolic syndromes remains debatable. Here, we

demonstrate that pyruvate dehydrogenase kinase 4 (PDK4) interacts with and stabilizes

IP3R1-GRP75-VDAC1 complex at the MAM interface. Obesity-induced increase in PDK4

activity augments MAM formation and suppresses insulin signaling. Conversely, PDK4

inhibition dampens MAM formation and improves insulin signaling by preventing MAM-

induced mitochondrial Ca2+ accumulation, mitochondrial dysfunction and ER stress.

Furthermore, Pdk4-/- mice exhibit reduced MAM formation and are protected against diet-

induced skeletal muscle insulin resistance. Finally, forced formation and stabilization of

MAMs with synthetic ER-mitochondria linker prevented the beneficial effects of PDK4

deficiency on insulin signaling. Overall, our findings demonstrate a critical mediatory role of

PDK4 in the development of skeletal muscle insulin resistance via enhancement of MAM

formation.

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INTRODUCTION

ER and mitochondria are distinct cellular organelles that are known to physically interact with each other to exchange phospholipids, calcium (Ca2+) and other metabolites to maintain cellular integrity and bioenergetics (1). Contact sites between ER and mitochondria are termed as mitochondria-associated ER membrane (MAM). MAM regulates Ca2+ transfer from ER to mitochondria via formation of a macromolecular complex comprising inositol 1,

4, 5-trisphosphate receptor type 1 (IP3R1), glucose-regulated protein 75 (GRP75), and voltage-dependent anion channel 1 (VDAC1) (2). IP3R1, an ER-resident Ca2+ release channel, is physically linked with the mitochondrial outer membrane protein VDAC1 via molecular chaperone GRP75. Several reports have shown that MAM plays a critical role in obesity- induced hepatic insulin resistance (IR) (3-5), although it is controversial whether the formation of MAMs prevents (3; 5) or promotes (4) IR. Given that skeletal muscle is a major site for regulating whole-body glucose homeostasis (6), these findings prompted us to investigate whether MAMs prevent or promote IR in skeletal muscle.

Although the molecular mechanisms underlying skeletal muscle IR are not fully understood, mitochondrial dysfunction has been implicated to play a key role in the development of this pathological condition (7). Accumulating evidence suggests that ER stress contributes to the development of IR (8). Recent studies have shown that deregulation in Ca2+ transfer from ER to mitochondria via MAM can cause ER stress and mitochondrial dysfunction (4; 5). For example, disruption of Ca2+ transfer between the two organelles by pharmacological and genetic inhibition of cyclophilin D led to ER stress and mitochondrial dysfunction in mouse hepatocytes and liver, resulting in the development of IR (5).

Furthermore, it was recently reported by the same group that disruption of SR/ER- mitochondria interaction in both skeletal muscle of obese mice and palmitate-treated

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myotubes is associated with IR, whereas enhancement of MAM prevents IR (9). However,

the findings we report here have led us to the opposite conclusion.

Pyruvate dehydrogenase kinases (PDKs) are mitochondrial enzymes that suppress the

conversion of pyruvate to acetyl CoA via inhibitory phosphorylation of the pyruvate

dehydrogenase complex (PDC) (10). To date, four isoenzymes of PDK (PDK1, 2, 3, and 4)

have been identified in mammals and are expressed in a tissue-specific manner (11). Among

them, PDK4 is predominantly expressed in skeletal muscle and heart. Previous studies have

shown that PDK4 expression is dramatically increased in skeletal muscle of insulin-resistant

patients and rodents (12; 13). High-fat diet (HFD)-challenged Pdk4-/- mice exhibit improved

glucose tolerance and insulin sensitivity (13; 14), suggesting that PDK4 may be a potential

therapeutic target against IR and its associated comorbidities (10). Although the beneficial

effects of PDK4 deficiency on IR are believed to be associated with PDC activation and

subsequent increase of glucose oxidation, the exact molecular mechanism remains unclear.

Recently, we have reported that PDK4 inhibition is closely associated with improvement of

mitochondrial function in several tissues during metabolic stress (15; 16). In addition, PDK4

was found in a proteomic study to be present in the MAM fraction of skeletal muscle (17).

Based on these findings, we speculated that PDK4 might play a role in the physical and/or

functional communication between SR/ER and mitochondria in skeletal muscle.

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MATERIALS AND METHODS

Animal models

Male 8 weeks-old wild type (WT) and Pdk4-/- C57Bl/6J mice (13) originated from the

Jackson Laboratory (Bar Harbor, ME, USA) were fed a control diet (CD) or 60% high fat diet (HFD) (D12492 Research Diets, Inc. New Brunswick, USA) and housed in a pathogen- free and temperature-controlled room (12:12 hour light dark cycle) for 16 weeks. Muscle tissues were harvested from these mice after 16 weeks on the diets and from male ob/ob mice under fed condition (Central Lab Animal Inc., South Korea) when they were 12 weeks old.

For insulin infusion, mice fasted for 16 h were injected with insulin (10mU/g body weight) intraperitoneally for 10 min. All experiments were performed in accordance with and approved by the Institutional Animal Care and Use Committee of Kyungpook National

University.

Cell lines

C2C12 cell line was purchased from ATCC (Manassas, VA, USA). WT and VXY/PDK4-flag expressing C2C12 myoblast were cultured in Dulbecco’s modified Eagle’s media (DMEM) high glucose media (#SH30243.01-Hyclone) supplemented with 10% FBS (#SH30084.03-

Hyclone, Australia), 1X Antibiotic-Antimycotic (#15240-062-Gibco). Cells were differentiated into myotubes with DMEM containing 2% horse serum (Gibco,NZ) for 5 days.

Primary skeletal muscle cells

Primary myotube and myoblast culture was performed with gastrocnemius muscle harvested from 8 weeks old WT or Pdk4-/- mice as described previously (18). siRNA, plasmids and adenovirus constructs siRNA targeting PDK4 (#1406883), GRP75 (#1370988), IP3R1 (#1374717), PDHE1α

(#1406708), Foxo1 (#1359213) and control siRNA (#SN-1003) were purchased from Bioneer

(Daejeon, South Korea). PDK4 kinase dead (ΔPDK4-flag) plasmid construct was designed

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by deleting the nucleic acid sequence that encodes aspartate and tryptophan DW (Asp394-

Trp395) motif as previously reported (19). D1ER and 4mitD3 plasmids were provided by Dr.

Kyu-Sang Park (Yonsei University, South Korea). Mitochondria-targeted blue fluorescence

protein (mito-BFP), mitochondria-targeted green fluorescence protein (mito-GFP), ER-

targeted Sec61B-GFP, and a similar synthetic ER-Mitochondria linker (Linker-RH) plasmid

used in previous reports (4; 20) was provided by Dr. Hyun-Woo Rhee (Seoul National

University, South Korea). Linker-RH carries mitochondria outer membrane (OMM) targeted

A kinase anchor protein 1 (AKAP1), Linker (20), red fluorescent protein (RFP) mCherry, ER

integral membrane protein phosphatidylinositide phosphatase Sac1 and hemagglutinin (HA)

encoding sequences to obtain 15-20nm space between ER and OMM. Vector backbone of all

the constructs is pcDNA3. PDK4 adenovirus was provided by Dr. Young-Bum Kim

(Harvard Medical School, USA). pcDNA and mock adenovirus served as control for

transient transfection and adenovirus transduction, respectively.

Transient transfection and adenovirus transduction

For myoblast, siRNA (100 nmol) or plasmid DNA was transfected using RNAi max reagent

(Thermofisher-13778150) or Lipofectamine2000 (Thermofisher-11668019), respectively and

cultured for 48 h. For myotubes, siRNA was transfected on the 3rd day of differentiation and

incubated for 2 days before any treatment. Adenovirus Linker-RH was transduced in C2C12

myotubes or primary myotubes for 48 h.

Experimental treatments

C2C12 myotubes were treated with BSA (Sigma-A7030) or BSA conjugated 0.4 mM

palmitate (Sigma-P9767) with and without 4 mM DCA (Sigma-Aldrich, St. Louis, MO) for

16 h. Thapsigargin (Sigma-T9033) 0.25µM or DMSO (Sigma-D2650) for various times. JNK

inhibitor SP600125 (ENZO Life Science) 20µM for 6 h. For insulin stimulation, the cells

were incubated in serum free medium containing 100 nM insulin (Sigma-I6634) for 10 min.

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In-situ Proximity Ligation assay (PLA)

In situ PLA in C2C12 cells were performed as described previously (3). For isolated single myofibers, myofibres were washed with PBS, fixed with 4% paraformaldehyde and permeabilized with 0.2% Triton-X in 0.1M glycine/PBS. Followed by blocking and overnight incubation with primary antibodies at 4oC. Myofibers were washed, probed with secondary antibodies conjugated with oligonucleotide, ligated and amplified using in-situ proximity ligation assay (PLA) kit (Duolink Sigma-DUO92101). Images were captured using Olympus

FluoView FV1000 confocal microscope or Olympus 1X81 fluorescence microscope

(Olympus Imaging, PA, USA). In situ PLA blob was counted using MetaMorph software

(Molecular Devices, LLC). Detailed antibody information is given in Table S1.

Immuno-fluorescence analysis (IF)

C2C12 myoblast were grown on coverslips, transfected with plasmid DNA, incubated for 24 h, washed with PBS, fixed with 4% paraformaldehyde, permeabilized and mounted with

VECTASHIELD mounting Medium (H1200). Images were captured using Olympus

FluoView FV1000 confocal microscope. Pearson co-efficient colocalization was analyzed by

ImageJ software with Fiji plugin (NIH, Bethesda, MD, USA).

Subcellular fractionation

Subcellular fractionation of C2C12 myotubes and gastrocnemius muscle (non-starved mice) was performed as previously described (21; 22).

Co-immunoprecipitation (co-IP) and Immunoblotting (IB)

Cells or tissues were lysed in lysis buffer (50mM Tris, 150mM Nacl, 1% Triton X-100, 1mM

EDTA and 10% glycerol) with protease (Roche-04693132001) and phosphatase inhibitors

(Sigma-P0044). 500µg protein lysate was incubated with GRP75 antibody and A/G plus agarose beads (Santacruz-2003) or anti-FLAG M2 Magnetic Beads (Sigma-M8823)

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overnight at 4oC. The solution was washed with lysis buffer and protein bound to the beads

was eluted with sample buffer and boiled.

For immunoblotting, cells or tissues were lysed in RIPA lysis buffer with protease and

phosphatase inhibitors. Protein samples were resolved by SDS-PAGE and transferred to

PVDF membranes. Detailed antibody information is given in Table S1. PDK3 and 4 anti-

serum were obtained from Dr. Robert Harris, Indiana University, USA. Intensity of

Immunoblot bands were quantified using ImageJ software.

Subcellular Ca2+ measurement

C2C12 cells were cultured on coverslips with Fura2-AM (Life Technology), washed with

Ca2+-free Krebs-Ringer bicarbonate (KRB) buffer, and transferred to a perfusion chamber on

an inverted microscope IX-70 (Olympus). The samples were alternately excited at 340 and

380 nm by a monochromatic light source LAMDA DG-4 (Sutter, Novato, CA, USA).

Fluorescence images were captured at 510 nm with an intensified CCD camera (Roper

Scientific, Trenton, NJ, USA). The two excitation wavelengths (F340/F380) fluorescence

intensity ratio was estimated by using MetaFluor 6.1 software (Molecular Devices, San Jose,

CA, USA). ER and mitochondrial Ca2+ were measured with FRET-based protein probe

D1ER and 4mitD3, respectively. Transfected cells were excited at 440 nm and YFP (540 nm)

CFP (490 nm) emission wave length monitored by the Nikon Ti-E inverted microscope

equipped with a Cascade 512B (EMCCD) camera (Roper). The ratiometric recording of

emitted fluorescence from YFP (540 nm) and CFP (490 nm) was created by MetaMorph

software (Molecular Devices).

Transmission electron microscopy (TEM)

Tissues and cells were fixed with 2.5 % glutaraldehyde, stained with 1% osmium tetroxide,

washed with PBS, dehydrated in ethanol and propylene oxide, embedded in Epon 812, and

polymerized. Sections were counterstained with uranyl acetate and lead citrate and visualized

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with a Bio-HVEM system (JEM-1400Plus at 120 kV and JEM-1000BEF at 1000 kV, JEOL,

JAPAN). ImageJ software was used for measuring the mitochondrial perimeter and surface area of MAM (distance between ER/SR and mitochondria within 30nm).

Mitochondrial ROS measurement

ROS was measured using ROS sensitive-MitoTracker red CM-H2XRos (Molecular probes-

M7513, Life technologies) and ROS-insensitive MitoTracker green FM (Molecular probes-

M7514, Life technologies) as previously described (23). The ratio between red and green fluorescence was quantified and analyzed using Image J software.

Mitochondrial membrane potential measurement (MMP)

MMP was assessed using JC-1 dye (Molecular probes, USA) by flow cytometer (FACS

Calibur; Becton Dickinson). JC-1 red/green ratio was analyzed using Cell Quest Pro. 4.0

Software.

ATP measurement

ATP was measured using ATPlite luminescence assay (Perkin Elmer-6016943) by following the manufacturer’s protocol and normalized with protein concentration.

Oxygen consumption rate (OCR)

OCR was measured using XF-24 flux analyzer as previously described (15).

Mitochondrial DNA quantification

Mitochondrial DNA (mtDNA) and genomic DNA (gDNA) were isolated with DNeasy Kit

(Qiagen-69504) and quantified by real-time PCR as previously described (24).

Statistical Analysis

Statistical significance was determined by the unpaired Student t test when two groups were compared. One or two-way analysis of variance (ANOVA) was used for comparisons among multiple groups. Values are presented as the mean ±SD of the indicated number of independent samples. P values <0.05 were considered statistically significant.

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RESULTS

PDK4 interacts with IP3R1-GRP75-VDAC1 complex at the MAM interface

We first validated the presence of the IP3R1-GRP75-VDAC1 complex at the MAM.

Subcellular fractions isolated from C2C12 myotubes (Figure S1A) or mouse gastrocnemius

muscle (Figure 1A) demonstrated the occurrence of these proteins in the MAM fraction. We

then evaluated the interactions between these proteins at the MAM interface via in situ PLA.

We observed that IP3R1, GRP75 and VDAC1 interact with each other in C2C12 myoblasts

(Figure S1B). These interactions were reconfirmed by co-IP in both C2C12 myotubes (Figure

S1C) and mouse gastrocnemius muscle (Figure 1C). Additionally, ryanodine receptor 1

(RyR1), a Ca2+ channel that also serves as a conduit for Ca2+ release from the SR/ER, was

detected in the MAM fraction (Figure 1A), but we did not observe any interactions between

RyR1 and GRP75 or VDAC1 (Figure S1B-C & 1C). We then examined the effects of

silencing IP3R1 or GRP75 on the interactions between IP3R1, GRP75, and VDAC1.

Knockdown of IP3R1 (Figure S1D) decreased the interaction between GRP75 and VDAC1

as well as its interactions with GRP75 or VDAC1 (Figure S1E). Likewise, knockdown of

GRP75 (Figure S1F) reduced the interaction between IP3R1 and VDAC1 as well as its

interactions with IP3R1 or VDAC1 (Figure S1G). Taken together, these findings suggest that

IP3R1, GRP75 and VDAC1 form a multiprotein complex at the MAM interface in skeletal

muscle.

We investigated whether PDK4 is present at the MAM interface in skeletal muscle

and C2C12 myotubes. PDK4 was detected abundantly in the mitochondrial fraction but not in

SR/ER fraction (Figure 1A & S1A). Interestingly, among the four PDK isoenzymes, only

PDK4 was detected in the MAM fraction (Figure 1A). To look for a potential role for PDK4

in MAM, we determined whether PDK4 interacts with the other MAM proteins by in situ

PLA with C2C12 myoblasts stably expressing PDK4-flag or VXY control. PDK4 clearly

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interacts with IP3R1, GRP75 and VDAC1 but not RyR1 (Figure 1B). This observation was further validated by co-IP, where GRP75 immuno-precipitated PDK4 along with VDAC1 and IP3R1 (Figure 1C). Similarly, co-IP with anti-flag antibody confirmed that PDK4 physically interacts with GRP75, IP3R1 and VDAC1 (Figure 1D).

We then investigated the subcellular site of the interaction between PDK4 and IP3R1,

GRP75 or VDAC1 by overlaying the in situ PLA blobs with mito-GFP. We observed that most of the interactions are in the vicinity of mitochondrial outer surface (Figure S1H). In addition, we transfected the PDK4-flag stable cells with Sec61b-GFP and mito-BFP to track

ER and mitochondria respectively (Figure 1E). The in situ PLA blobs of PDK4-flag/IP3R1 as well as IP3R1-VDAC1 were localized at the ER-mitochondria interface, as visualized by confocal microscope (Figure 1F). Taken together, these results indicated that PDK4 interacts with IP3R1-GRP75-VDAC1 complex at the MAM interface.

PDK4 kinase activity is crucial for MAM formation

PDK4 is a serine/threonine kinase. We examined whether its kinase activity is required for interacting with the components of the IP3R1 channeling complex. To this end, we performed in situ PLA (Figure 2A-B) and co-IP (Figure 2C-E) in PDK4-flag overexpressing C2C12 cells in the absence or presence of dichloroacetate (DCA), a known synthetic inhibitor of PDKs. DCA treatment suppressed the interaction of PDK4 with IP3R1,

GRP75, or VDAC1 (Figure 2A, C-E). In addition, enhanced IP3R1-GRP75-VDAC1 interactions induced by PDK4-flag overexpression were markedly reduced by DCA treatment

(Figure 2B). To be more specific, we constructed a kinase activity dead PDK4 (∆PDK4-flag) mutant. Overexpression of ∆PDK4-flag failed to induce PDHE1 (a component of PDC) phosphorylation at serine 293 and 300 residues (Figure S2A), confirming the loss of its kinase activity. Unlike PDK4-flag, ∆PDK4-flag overexpression failed to induce IP3R1-

GRP75-VDAC1 interactions (Figure 2F). Moreover, we did not observe any interaction

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between PDHE1 and IP3R1 or VDAC1 (interaction between PDHE1 and PDK2 in the

absence and presence of siPDHE1 was used as negative and positive controls, respectively)

(Figure S2B). Additionally, siRNA-mediated knockdown of PDHE1 did not affect the

IP3R1-GRP75-VDAC1 interactions in the basal and PDK4 overexpressed condition (Figure

S2C-E). Taken together, these results suggest that the PDK4 promotes IP3R1-GRP75-

VDAC1 complex formation independent of PDC activity.

Obesity augments MAM formation in skeletal muscle

Next, we examined the role of PDK4 in MAM formation in the context of obesity-

induced skeletal muscle IR. To understand the role of MAM formation in obesity, mice were

sacrifice in fed state to rule out the independent effects of fasting on MAM formation. As

previously reported (13), we observed a robust increase in the PDK4 protein level, but not of

PDK1, 2 or 3 in skeletal muscle of HFD fed and genetically obese, ob/ob mice compared to

CD fed and lean mice, respectively (Figure 3A-B). Increased plasma free fatty acids level,

especially palmitate, is associated with obesity and lipotoxicity and plays a critical role in the

development of obesity-induced IR (25). Similarly, palmitate-treated C2C12 myotubes show

elevated PDK4 protein level but not of other isoenzymes (Figure S3A-B). Interestingly, we

observed a significant enrichment of PDK4 along with other known MAM proteins IP3R1,

GRP75 and VDAC1 in the MAM fraction of HFD-fed mice (Figure 3C) or upon palmitate

treatment (Figure S3C). Moreover, IP3R1-GRP75- VDAC1 interactions were significantly

increased in the myofibers isolated from HFD-fed mice compared to CD-fed mice, as

visualized by in situ PLA (Figure 3D). Additionally, the MAM fraction isolated from ob/ob

mice has increased PDK4, IP3R1, GRP75 and VDAC1 protein levels compared to lean mice

(Figure 3E). In situ PLA in myofibers isolated from ob/ob mice also showed enhanced IP3R-

GRP75-VDAC1 interactions (Figure 3F). Altogether, these findings indicate enhanced MAM

formation in skeletal muscle during obesity.

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To investigate the effect of palmitate on the integrity of IP3R1 channeling complex, we monitored IP3R1-VDAC1 interaction by in situ PLA. Palmitate treatment increases

IP3R1-VDAC1 interaction along with a concomitant increase in PDK4 protein level (Figure

S3D-E and S3H). Additionally, we also evaluated whether glucose has any synergistic effect on palmitate-induced IP3R1-VDAC1 interaction. However, we did not observe any significant effect of glucose at either low or high concentration on palmitate-induced dose- dependent increase in IP3R1-VDAC1 interaction (Figure S3F-G). Several transcription factors including PPARα, PPARγ, PGC1α, and FoxO1 have been reported to regulate PDK4 expression (26). Among them, dephosphorylation and activation of FoxO1 is closely associated with PDK4 induction in vitro and in vivo (Figure S3H-I). Palmitate-induced upregulation of PDK4 was reversed by FoxO1 siRNA, but not of PDK2 (Figure S3J).

Furthermore, co-IP assay demonstrated that the interaction of PDK4 with IP3R1, GRP75 or

VDAC1 was significantly increased upon palmitate treatment (Figure 3G-I). Taken together, these results indicate that PDK4 enrichment in the MAM during obesity is associated with enhanced mitochondria-ER/SR interactions in the skeletal muscle of obese mice.

PDK4 ablation hinders MAM formation in diet-induced obese mice skeletal muscle

Next, we examined whether genetic ablation of PDK4 could suppress MAM formation in diet-induced obese mice. To evaluate this, we isolated subcellular fractions from gastrocnemius muscle of WT and Pdk4-/- mice fed with CD or HFD. We observed that expression level of MAM-resident proteins in total homogenates from skeletal muscle of

Pdk4-/- mice were comparable to that in WT mice. In addition, HFD feeding did not affect their expression levels in WT or Pdk4-/- mice (Figure S4A-B). However, HFD-induced enrichment of these proteins in the MAM fraction from skeletal muscle of WT mice was reduced in Pdk4-/- mice (Figure 4A-B). Moreover, in situ PLA of myofibers isolated from gastrocnemius muscle of HFD-fed Pdk4-/- mice exhibited a reduction in IP3R1-VDAC1-

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GRP75 interactions compared to HFD-fed WT mice (Figure 4C), indicating a reduction in

MAM formation. Similarly, palmitate-induced increase in IP3R1-GRP75 or VDAC1

interactions and enrichment of IP3R1 in crude mitochondrial fraction (MAM containing

mitochondrial extract) were significantly decreased upon PDK4 knockdown (Figure S4C-E).

Furthermore, transmission electron microscopy (TEM) analysis of gastrocnemius muscle

showed significantly increased intermyofibrillar (IMF) MAM (SR/ER and mitochondria

contacts within 30nm distance) surface area in HFD-fed WT mice compared to controls.

Pdk4-/- mice significantly reversed this effect (Figure 4D-E). In addition, the distance

between SR/ER and mitochondria in MAM was taken into account considering the irregular

distance (0~30nm) observed in TEM images. A distance (>15nm) between SR/ER and

mitochondria, which is considered to be loose MAM, is required for accommodating IP3R1-

VDAC1-GRP75 complex formation and transfer Ca2+ efficiently whereas, below 15nm (tight

MAM) distance is considered unsuitable for Ca2+ transfer (27). Interestingly, we observed an

increase in loose MAM and reduction in tight MAM percentage in total MAM surface area in

HFD-fed WT mice compared to controls or HFD-fed Pdk4-/- mice (Figure 4F). However,

MAM was not observed in subsarcolemmal (SS) mitochondria (Figure S4F). Similarly,

population of ER-associated mitochondria and MAM surface area were significantly

increased in palmitate treated WT myoblasts and this effect was reduced in Pdk4-/- myoblasts

(Figure S4G-I). Next, we asked whether deficiency of PDK4 has any effect on basal

mitochondrial dynamics that might negatively affect the structural integrity of MAM. To this

end, we evaluated the expression level of proteins that are involved in mitochondrial fusion

and fission machinery. PDK4 deficiency had no effect on the expression level of fusion

proteins Mfn1, Mfn2, OPA1 or fission proteins Fis1, Drp1 and its regulatory phosphorylation

sites (s616 and s637) (Figure S4J-K and S4A-B). Moreover, we did not observe any

significant difference in total, intermyofibrillar (IMF) and subsarcolemmal (SS)

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mitochondrial perimeter between WT and Pdk4-/- skeletal muscle (Figure S4L). In addition, basal mitochondria oxygen consumption rate (OCR) was similar between WT and Pdk4-/- myotubes (Figure S4M-N). Altogether, these results suggest that PDK4 deficiency reduces

MAM formation in skeletal muscle under metabolic stress.

PDK4 inhibition attenuates Ca2+ transfer from ER to mitochondria, mitochondrial dysfunction and ER stress

MAM plays a key role in regulating Ca2+ transfer from ER to mitochondria via formation of IP3R1 channeling complex (2). Previous studies have demonstrated that metabolic stress enhances Ca2+ transfer from ER to mitochondria, resulting in mitochondrial

Ca2+ overload (4; 28). Therefore, to determine the role of PDK4 in palmitate-induced Ca2+ transfer between the two organelles, we measured mitochondrial Ca2+ levels in C2C12 myoblasts using a mitochondria specific Ca2+ probe, 4mitD3 (29). As expected, the basal

2+ [Ca ]mito levels were significantly higher in palmitate-treated cells compared to vehicle.

2+ However, co-treatment of DCA lowered basal [Ca ]mito level compared to palmitate-

2+ treatment (Figure 5A, i-ii). In addition, we detected higher peak of [Ca ]mito following stimulation of IP3R-mediated ER Ca2+ release by ATP (30) upon palmitate treatment. These effects of palmitate were attenuated in the presence of DCA (Figure 5A, i & iii). We next

2+ 2+ measured [Ca ]ER levels using D1ER, an ER specific Ca probe (31), after stimulating with vehicle, palmitate, or palmitate/DCA. Both basal and cyclopiazonic acid (CPA; SERCA inhibitor)-induced ER Ca2+ depletion rate was similar in all groups, suggesting that PDK4 inhibition does not affect the ER Ca2+ store or release machinery (Figure 5B, i-iii).

2+ 2+ Additionally, we also monitored [Ca ]cyto using FURA-2, a cytosolic Ca indicator but failed to notice any significant difference among the groups (Figure 5C, i-ii). However,

2+ 2+ draining out of [Ca ]ER by CPA in palmitate-treated cells released less Ca to cytosol, thereby suggesting that major portion of the ER Ca2+ goes to mitochondria due to enhanced

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MAM formation. This effect was reversed upon PDK4 inhibition by DCA (Figure 5C, i & iii).

Taken together, these results indicate that suppression of MAM formation by PDK4

inhibition decreases palmitate-induced Ca2+ transfer from ER to mitochondria.

Enhanced Ca2+ transfer from ER to mitochondria via MAM can cause ER stress and

mitochondrial dysfunction (4; 32). Thus, we investigated whether disruption of MAM could

attenuate ER stress and mitochondrial dysfunction in skeletal muscle in the context of obesity.

Palmitate-induced mitochondrial reactive oxygen species (ROS) generation was significantly

suppressed by silencing PDK4 as well as components of IP3R1 channeling complex such as

GRP75 or IP3R1 (Figure 5D and S5A). As previously reported (33), palmitate treatment

resulted in a reduction of mitochondrial membrane potential (MMP). These effects were

reversed upon knockdown of PDK4 (Figure 5E). Furthermore, palmitate-induced decrease in

ATP production was also reversed by silencing PDK4 or other MAM components GRP75 or

IP3R1 (Figure 5F and S5B). Mitochondrial density and activity play a crucial role in skeletal

muscle function (34). Interestingly, deficiency of PDK4 prevented the depletion of

mitochondrial DNA (mtDNA) copy number in HFD-fed mice (Figure 5G). However, the

mitochondrial OXPHOS-complex protein level was similar between the groups (Figure S5C-

D).

Next, we asked whether disruption of MAM could attenuate obesity-induced ER stress. Both

HFD or palmitate-induced ER stress as observed by increased IRE1a, eIF2a phosphorylation

and CHOP expression was markedly suppressed in Pdk4-/- mice (Figure 5H-I) or upon PDK4

knockdown (Figure 5J-K). Similar results were obtained upon IP3R1 or GRP75 silencing

(Figure S5E-F), suggesting that disruption of MAM formation attenuates palmitate-induced

ER stress.

ER stress inducer Thapsigargin (Tg), a SERCA inhibitor, enhances ER Ca2+ depletion and

induces mitochondria Ca2+ dependent apoptosis (35). Tg-induced ER stress and IP3R1-

17 Diabetes Page 18 of 91

VDAC1 interaction was reversed upon PDK4 knockdown (Figure S5G-L). We also observed that disruption of IP3R1 channeling complex by GRP75 knockdown suppressed Tg-induced

CHOP expression whereas, enhancement of IP3R1 channeling by PDK4 overexpression further amplified Tg-induced CHOP expression. However, this effect of PDK4 overexpression was suppressed by knockdown of GRP75 (Figure S5M). Taken together, these results suggest that PDK4 inhibition suppresses induction of ER stress and mitochondrial dysfunction via downregulation of MAM formation (Figure S5N).

PDK4 ablation attenuates obesity-induced skeletal muscle IR via downregulation of

MAM

ER stress mediated JNK activation plays a critical role in the pathogenesis of IR by phosphorylating IRS1 at serine 307 which subsequently impairs insulin-induced AKT signaling pathway in skeletal muscle (36). Thus, we examined the effect of JNK1 activation on MAM formation. We observed that palmitate-induced JNK1 activation and inhibition of insulin-induced AKT phosphorylation was reversed upon knockdown of PDK4 (Figure 6A-

B), suggesting PDK4 knockdown attenuates palmitate-induced impairment of insulin signaling. Similarly, knockdown of IP3R1 or GRP75 reduced JNK1 activation in palmitate- treated cells (Figure S6A & C) and restored insulin-stimulated AKT signaling (Figure S6B &

D). Furthermore, HFD-induced JNK1 activation and inhibition of insulin-induced AKT phosphorylation was markedly reversed in gastrocnemius muscle of Pdk4-/- mice. In addition, inhibitory phosphorylation of IRS-1 at serine 307 was significantly decreased in gastrocnemius muscle of HFD-fed Pdk4-/- mice compared to WT mice (Figure 6C-D). Taken together, these results suggest that genetic ablation of PDK4 dampens MAM formation and obesity-induced skeletal muscle IR via suppression of JNK activation.

Finally, to demonstrate the causal relationship between PDK4 and MAM in IR, we asked whether the enhancement of MAM formation could reverse the effects of PDK4

18 Page 19 of 91 Diabetes

deficiency on palmitate-induced IR. To this end, we utilized a synthetic ER-mitochondria

linker (linker-RH) to artificially enhance SR/ER-mitochondria interaction (Figure 6E). The

linker-RH expression significantly increased ER-mitochondria interaction and mitochondrial

(both basal and stimulated) Ca2+ levels (Figure 6F-H). Linker-RH expression induced JNK1

phosphorylation and attenuated insulin-induced AKT phosphorylation in C2C12 myotubes

(Figure S6E-F), suggesting that an increase in MAM formation is sufficient to trigger IR.

Furthermore, we examined whether PDK4 deficiency could reverse the effect of the linker-

RH. Surprisingly, deficiency of PDK4 failed to suppress linker-mediated JNK1 activation

and insulin-induced AKT phosphorylation (Figure 6I-J & S6G-H), indicating that MAM

formation occurs downstream of PDK4 signaling. Interestingly, JNK inhibitor-SP600125

treatment significantly reversed the inhibitory effect of linker-RH expression on AKT

phosphorylation (Figure 6K-L), thereby indicating that the linker-mediated induction of IR is

dependent on JNK activation. Altogether, we conclude that PDK4 inhibition improves insulin

signaling via suppression of MAM formation in skeletal muscle.

19 Diabetes Page 20 of 91

DISCUSSION

ER and mitochondria crosstalk via MAM is believed to play a critical role in the maintenance of cellular homeostasis. Dysregulation of MAM is implicated in several human diseases (37). The MAM-resident multi-protein complex of IP3R1-GRP75-VDAC1 regulates

Ca2+ transport from the ER to the mitochondria. Previous reports have demonstrated the importance of this multi-protein complex and its association with hepatic IR (3; 4). However, skeletal muscle being a major tissue affected during IR, the role of MAM and its regulation in the context of the IR remained unclear. Here we have found that a PDK4 subpopulation resides at MAM and directly interacts with IP3R1-GRP75-VDAC1 complex to regulate obesity-induced MAM formation, thereby implicating a key regulatory role of PDK4 in the pathogenesis of skeletal muscle IR (Figure 7). RyR1 is another type of SR/ER Ca2+ release channel which is highly expressed in skeletal muscle (38). Because we observed the presence of RyR1 in the MAM, we speculated that RyR1 might be an interacting partner of GRP75 and VDAC1 to regulate Ca2+ transfer from SR/ER to mitochondria at the MAM interface.

However, we did not observe such interactions. It was previously shown that the cross-talk between IP3R1 and RyR1 contributes to Ca2+ spark in skeletal muscle (39; 40). Thus, we speculate that the presence of RyR1 in the MAM might amplify Ca2+ release from SR/ER in response to IP3R1-mediated local Ca2+ spark. Further study is required to explore the potential cross-talk between IP3R1 channeling complex and RyR1 in the MAM.

Pharmacological inhibition of PDK activity with DCA and experiments with a PDK4 kinase-deleted mutant demonstrated that kinase activity of PDK4 plays an essential role in the regulation of MAM formation. Interestingly, we have found that knockdown of PDHE1, the substrate for PDK4 in the PDC (10), did not affect the integrity of IP3R1-GRP75-VDAC1 complex, suggesting that regulation of MAM formation by PDK4 is independent of PDC.

Although target substrates for PDK4 other than PDHE1α have been not firmly established,

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we speculate that PDK4 might be able to phosphorylate one of these interacting partners in

MAM; a hypothesis that warrants future investigation. Furthermore, it can be hypothesized

that there are two subpopulations of PDK4, one regulating PDC complex in mitochondrial

matrix to suppress pyruvate oxidation, whereas the second subpopulation is involved in

MAM formation and Ca2+ transport to mitochondria, thereby contributing towards fatty acid

metabolism (via TCA cycle). Mitochondrial Ca2+ is a key molecule that drives TCA cycle

and ATP synthesis by stimulating isocitrate dehydrogenase, alpha-keto glutarate

dehydrogenase and ATP synthase (41). We showed that palmitate induces IP3R1-VDAC1

interaction in a time dependent manner. As described in previous reports (28; 42), it is very

likely that in the early phase, MAM formation might be beneficial to boost fatty acid

oxidation and ATP production but sustained MAM formation induced by palmitate leads to a

constant rise in mitochondrial Ca2+ level, which subsequently leads to increased ROS

generation and mitochondrial dysfunction. In this context, previous studies demonstrated

opposing effects of short term (43) versus prolonged exposure (44) of palmitate on fatty acid

oxidation. Mitochondrial ROS is known to trigger ER stress (32). Therefore, our findings

suggest that PDK4 inhibition prevents fatty acid-induced mitochondrial Ca2+ accumulation

and ROS generation, leading to improvement of mitochondrial dysfunction and ER stress via

suppression of MAM formation.

Role of MAM in obesity-induced IR has been investigated in hepatic tissue but it is

still controversial due to contradictory lines of evidence (3-5). Here, we have observed that

MAM is markedly increased in skeletal muscle of both diet and genetically-induced obesity.

Our findings are consistent with a previous study showing that excessive ER-mitochondrial

coupling contributes to the development of hepatic IR (4). Recently, and contradictory to our

current findings, Tubbs et al. (9) reported that MAM formation in skeletal muscle is disrupted

as a consequence of diet-induced obesity in mice. We suspect that the discrepancy is due to a

21 Diabetes Page 22 of 91

major difference in diets used in the studies. The high fat high sucrose diet (HFHSD) used by

Tubbs et al. is highly inflammatory and rapidly alters muscle integrity compared to HFD diet used in our study (45; 46). In addition, a method adopted by Tubbs et al. for quantifying the

MAM amount by isolation of MAM fraction is not a robust method when comparing healthy and unhealthy groups. HFHSD skeletal muscle tissue induces mitochondrial damage (47), therefore there is a high chance of losing the MAM population associated with the damaged mitochondria during subcellular fractionation process. Therefore, quantifying the protein concentration from isolated MAM homogenates to determine the MAM amount might give inaccurate information. We also cannot rule out that the differences in species (human versus mouse), cell culture conditions and time periods chosen for analysis may result in different findings as MAM is highly dynamic in response to environmental factors and cellular status

(40). Furthermore, our findings agree well with the finding that patients with non-alcoholic steatohepatitis have increased expression of IP3R1 and the amount of MAM correlated with the degree of steatosis in human liver biopsies (48). Thus, we postulate that the discrepancy between these two studies may be explained primarily by difference in diets but also methodology and experimental conditions.

JNK is known to mediate IR under conditions of mitochondrial dysfunction and ER stress (49). Previously, JNK1 deficiency has been shown to prevent diet-induced IR in skeletal muscle (50). We observed that deficiency of PDK4 and other MAM components suppressed JNK1 activation to ameliorate IR. Consistent with these findings, we recently reported that cisplatin-induced JNK activation is suppressed by pharmacological inhibition or genetic ablation of PDK4 (16). We also found that artificial enhancement of ER- mitochondria coupling using a synthetic ER-mitochondria linker triggered JNK1 activation and reduced insulin-induced AKT activation in myotubes. These detrimental effects of linker overexpression were reversed by pharmacological inhibition of JNK, suggesting that linker-

22 Page 23 of 91 Diabetes

mediated IR is dependent on JNK activation. This is in line with a previous study showing

that artificial enrichment of ER-mitochondrial contacts instigates obesity-induced hepatic IR

via a JNK-dependent mechanism (4).

Overall, we conclude that PDK4 deficiency leads to destabilization of IP3R1-GRP75-

VDAC1 complex which subsequently leads to the suppression of JNK activation and protects

from skeletal muscle IR in the context of obesity. Our data provide important evidence that

MAM is associated with skeletal muscle IR and identifies PDK4 as a novel modulator of

MAM integrity. Our study strengthens the notion that modulating MAM formation could be a

promising strategy for the treatment of obesity-related metabolic diseases.

23 Diabetes Page 24 of 91

ACKNOWLEDGMENTS

This work was supported by the National Research Foundation of Korea (NRF) grant funded by the Korean government (MSIP) (NRF-2016M3A9B6902872, NRF-2017R1A2B3006406,

NRF-2016R1E1A2020567, and NRF-2017R1E1A2A02023467) and a grant of the Korea

Health technology R&D Project through the Korea Health Industry Development Institute

(KHIDI), funded by the Ministry of Health & Welfare, Republic of Korea (Grant Number:

HI16C1501) and a grant funded by Korea Basic Science Institute (Grant Number: T38210) and VA Merit Award 1I01CX000361, NIH U01AA021840, NIH R01 DK107682, NIH R01

AA025208, US DOD W81XWH-12-1-0497 and NIH R21AA024935-01.

AUTHOR CONTRIBUTIONS

T.T. performed experiments, and analyzed and interpreted data. T.T., J.L., and I-K.L. designed experiments, discussed data, and wrote the manuscript. C-M.H., J-S. P., H-J.K.,

Y.H.H., K-S.P. contributed to experiments and data collection. J-H.J., L.S., T-H.K., K-G.P.,

R.A.H., SL., Y.H.H., K-S.P. provided reagents, materials and discussed data. T.T., D.C., SL.,

R.A.H, and I-K.L., edited and revised manuscript. I-K.L. supervised the work.

DECLARATION OF INTERESTS

All authors declare no conflict of interest.

24 Page 25 of 91 Diabetes

REFERENCES

1. Arruda AP, Hotamisligil GS: Calcium Homeostasis and Organelle Function in the Pathogenesis of Obesity and Diabetes. Cell Metab 2015;22:381-397 2. Szabadkai G, Bianchi K, Varnai P, De Stefani D, Wieckowski MR, Cavagna D, Nagy AI, Balla T, Rizzuto R: Chaperone-mediated coupling of endoplasmic reticulum and mitochondrial Ca2+ channels. J Cell Biol 2006;175:901-911 3. Tubbs E, Theurey P, Vial G, Bendridi N, Bravard A, Chauvin MA, Ji-Cao J, Zoulim F, Bartosch B, Ovize M, Vidal H, Rieusset J: Mitochondria-associated endoplasmic reticulum membrane (MAM) integrity is required for insulin signaling and is implicated in hepatic insulin resistance. Diabetes 2014;63:3279-3294 4. Arruda AP, Pers BM, Parlakgul G, Guney E, Inouye K, Hotamisligil GS: Chronic enrichment of hepatic endoplasmic reticulum-mitochondria contact leads to mitochondrial dysfunction in obesity. Nat Med 2014;20:1427-1435 5. Rieusset J, Fauconnier J, Paillard M, Belaidi E, Tubbs E, Chauvin MA, Durand A, Bravard A, Teixeira G, Bartosch B, Michelet M, Theurey P, Vial G, Demion M, Blond E, Zoulim F, Gomez L, Vidal H, Lacampagne A, Ovize M: Disruption of calcium transfer from ER to mitochondria links alterations of mitochondria-associated ER membrane integrity to hepatic insulin resistance. Diabetologia 2016;59:614-623 6. Bouzakri K, Koistinen HA, Zierath JR: Molecular mechanisms of skeletal muscle insulin resistance in type 2 diabetes. Curr Diabetes Rev 2005;1:167-174 7. Gonzalez-Franquesa A, Patti ME: Insulin Resistance and Mitochondrial Dysfunction. Advances in experimental medicine and biology 2017;982:465-520 8. Salvado L, Palomer X, Barroso E, Vazquez-Carrera M: Targeting endoplasmic reticulum stress in insulin resistance. Trends Endocrinol Metab 2015;26:438-448 9. Tubbs E, Chanon S, Robert M, Bendridi N, Bidaux G, Chauvin MA, Ji-Cao J, Durand C, Gauvrit-Ramette D, Vidal H, Lefai E, Rieusset J: Disruption of Mitochondria-Associated Endoplasmic Reticulum Membranes (MAMs) Integrity Contributes to Muscle Insulin Resistance in Mice and Humans. Diabetes 2018; 10. Zhang S, Hulver MW, McMillan RP, Cline MA, Gilbert ER: The pivotal role of pyruvate dehydrogenase kinases in metabolic flexibility. Nutr Metab (Lond) 2014;11:10 11. Bowker-Kinley MM, Davis WI, Wu P, Harris RA, Popov KM: Evidence for existence of tissue-specific regulation of the mammalian pyruvate dehydrogenase complex. Biochem J 1998;329 ( Pt 1):191-196 12. McAinch AJ, Cornall LM, Watts R, Hryciw DH, O'Brien PE, Cameron-Smith D: Increased pyruvate dehydrogenase kinase expression in cultured myotubes from obese and diabetic individuals. Eur J Nutr 2015;54:1033-1043 13. Jeoung NH, Harris RA: Pyruvate dehydrogenase kinase-4 deficiency lowers blood glucose and improves glucose tolerance in diet-induced obese mice. Am J Physiol Endocrinol Metab 2008;295:E46-54 14. Hwang B, Jeoung NH, Harris RA: Pyruvate dehydrogenase kinase isoenzyme 4 (PDHK4) deficiency attenuates the long-term negative effects of a high-saturated fat diet. Biochem J 2009;423:243-252 15. Lee SJ, Jeong JY, Oh CJ, Park S, Kim JY, Kim HJ, Doo Kim N, Choi YK, Do JY, Go Y, Ha CM, Choi JY, Huh S, Ho Jeoung N, Lee KU, Choi HS, Wang Y, Park KG, Harris RA, Lee IK: Pyruvate Dehydrogenase Kinase 4 Promotes Vascular Calcification via SMAD1/5/8 Phosphorylation. Sci Rep 2015;5:16577 16. Oh CJ, Ha CM, Choi YK, Park S, Choe MS, Jeoung NH, Huh YH, Kim HJ, Kweon HS, Lee JM, Lee SJ, Jeon JH, Harris RA, Park KG, Lee IK: Pyruvate

25 Diabetes Page 26 of 91

dehydrogenase kinase 4 deficiency attenuates cisplatin-induced acute kidney injury. Kidney Int 2017;91:880-895 17. Liu Z, Du X, Deng J, Gu M, Hu H, Gui M, Yin CC, Chang Z: The interactions between mitochondria and sarcoplasmic reticulum and the proteome characterization of mitochondrion-associated membrane from rabbit skeletal muscle. Proteomics 2015;15:2701-2704 18. Pasut A, Jones AE, Rudnicki MA: Isolation and culture of individual myofibers and their satellite cells from adult skeletal muscle. Journal of visualized experiments : JoVE 2013:e50074 19. Wynn RM, Kato M, Chuang JL, Tso SC, Li J, Chuang DT: Pyruvate dehydrogenase kinase-4 structures reveal a metastable open conformation fostering robust core-free basal activity. J Biol Chem 2008;283:25305-25315 20. Csordas G, Renken C, Varnai P, Walter L, Weaver D, Buttle KF, Balla T, Mannella CA, Hajnoczky G: Structural and functional features and significance of the physical linkage between ER and mitochondria. J Cell Biol 2006;174:915-921 21. Wieckowski MR, Giorgi C, Lebiedzinska M, Duszynski J, Pinton P: Isolation of mitochondria-associated membranes and mitochondria from animal tissues and cells. Nat Protoc 2009;4:1582-1590 22. Frezza C, Cipolat S, Scorrano L: Organelle isolation: functional mitochondria from mouse liver, muscle and cultured fibroblasts. Nat Protoc 2007;2:287-295 23. Kuznetsov AV, Kehrer I, Kozlov AV, Haller M, Redl H, Hermann M, Grimm M, Troppmair J: Mitochondrial ROS production under cellular stress: comparison of different detection methods. Analytical and bioanalytical chemistry 2011;400:2383- 2390 24. Sakellariou GK, Pearson T, Lightfoot AP, Nye GA, Wells N, Giakoumaki, II, Vasilaki A, Griffiths RD, Jackson MJ, McArdle A: Mitochondrial ROS regulate oxidative damage and mitophagy but not age-related muscle fiber atrophy. Sci Rep 2016;6:33944 25. Yazici D, Sezer H: Insulin Resistance, Obesity and Lipotoxicity. Advances in experimental medicine and biology 2017;960:277-304 26. Jeong JY, Jeoung NH, Park KG, Lee IK: Transcriptional regulation of pyruvate dehydrogenase kinase. Diabetes Metab J 2012;36:328-335 27. Giacomello M, Pellegrini L: The coming of age of the mitochondria-ER contact: a matter of thickness. Cell Death Differ 2016;23:1417-1427 28. Egnatchik RA, Leamy AK, Jacobson DA, Shiota M, Young JD: ER calcium release promotes mitochondrial dysfunction and hepatic cell lipotoxicity in response to palmitate overload. Mol Metab 2014;3:544-553 29. Palmer AE, Tsien RY: Measuring calcium signaling using genetically targetable fluorescent indicators. Nat Protoc 2006;1:1057-1065 30. Henning RH, Duin M, van Popta JP, Nelemans A, den Hertog A: Different mechanisms of Ca2(+)-handling following nicotinic acetylcholine receptor stimulation, P2U-purinoceptor stimulation and K(+)-induced depolarization in C2C12 myotubes. Br J Pharmacol 1996;117:1785-1791 31. Palmer AE, Jin C, Reed JC, Tsien RY: Bcl-2-mediated alterations in endoplasmic reticulum Ca2+ analyzed with an improved genetically encoded fluorescent sensor. Proc Natl Acad Sci U S A 2004;101:17404-17409 32. Ly LD, Xu S, Choi SK, Ha CM, Thoudam T, Cha SK, Wiederkehr A, Wollheim CB, Lee IK, Park KS: Oxidative stress and calcium dysregulation by palmitate in type 2 diabetes. Exp Mol Med 2017;49:e291

26 Page 27 of 91 Diabetes

33. Jheng HF, Tsai PJ, Guo SM, Kuo LH, Chang CS, Su IJ, Chang CR, Tsai YS: Mitochondrial fission contributes to mitochondrial dysfunction and insulin resistance in skeletal muscle. Mol Cell Biol 2012;32:309-319 34. Porter C, Wall BT: Skeletal muscle mitochondrial function: is it quality or quantity that makes the difference in insulin resistance? J Physiol 2012;590:5935-5936 35. Zhang D, Armstrong JS: Bax and the mitochondrial permeability transition cooperate in the release of cytochrome c during endoplasmic reticulum-stress- induced apoptosis. Cell Death Differ 2007;14:703-715 36. Solinas G, Becattini B: JNK at the crossroad of obesity, insulin resistance, and cell stress response. Mol Metab 2017;6:174-184 37. van Vliet AR, Verfaillie T, Agostinis P: New functions of mitochondria associated membranes in cellular signaling. Biochim Biophys Acta 2014;1843:2253-2262 38. Yi M, Weaver D, Eisner V, Varnai P, Hunyady L, Ma J, Csordas G, Hajnoczky G: Switch from ER-mitochondrial to SR-mitochondrial calcium coupling during muscle differentiation. Cell Calcium 2012;52:355-365 39. Tjondrokoesoemo A, Li N, Lin PH, Pan Z, Ferrante CJ, Shirokova N, Brotto M, Weisleder N, Ma J: Type 1 inositol (1,4,5)-trisphosphate receptor activates ryanodine receptor 1 to mediate calcium spark signaling in adult mammalian skeletal muscle. J Biol Chem 2013;288:2103-2109 40. Eisner V, Csordas G, Hajnoczky G: Interactions between sarco-endoplasmic reticulum and mitochondria in cardiac and skeletal muscle - pivotal roles in Ca(2)(+) and reactive oxygen species signaling. J Cell Sci 2013;126:2965-2978 41. Tarasov AI, Griffiths EJ, Rutter GA: Regulation of ATP production by mitochondrial Ca(2+). Cell Calcium 2012;52:28-35 42. Bravo R, Vicencio JM, Parra V, Troncoso R, Munoz JP, Bui M, Quiroga C, Rodriguez AE, Verdejo HE, Ferreira J, Iglewski M, Chiong M, Simmen T, Zorzano A, Hill JA, Rothermel BA, Szabadkai G, Lavandero S: Increased ER-mitochondrial coupling promotes mitochondrial respiration and bioenergetics during early phases of ER stress. J Cell Sci 2011;124:2143-2152 43. Samovski D, Sun J, Pietka T, Gross RW, Eckel RH, Su X, Stahl PD, Abumrad NA: Regulation of AMPK activation by CD36 links fatty acid uptake to beta-oxidation. Diabetes 2015;64:353-359 44. Salvado L, Coll T, Gomez-Foix AM, Salmeron E, Barroso E, Palomer X, Vazquez-Carrera M: Oleate prevents saturated-fatty-acid-induced ER stress, inflammation and insulin resistance in skeletal muscle cells through an AMPK- dependent mechanism. Diabetologia 2013;56:1372-1382 45. Ishimoto T, Lanaspa MA, Rivard CJ, Roncal-Jimenez CA, Orlicky DJ, Cicerchi C, McMahan RH, Abdelmalek MF, Rosen HR, Jackman MR, MacLean PS, Diggle CP, Asipu A, Inaba S, Kosugi T, Sato W, Maruyama S, Sanchez-Lozada LG, Sautin YY, Hill JO, Bonthron DT, Johnson RJ: High-fat and high-sucrose (western) diet induces steatohepatitis that is dependent on fructokinase. Hepatology 2013;58:1632-1643 46. Collins KH, Paul HA, Hart DA, Reimer RA, Smith IC, Rios JL, Seerattan RA, Herzog W: A High-Fat High-Sucrose Diet Rapidly Alters Muscle Integrity, Inflammation and Gut Microbiota in Male Rats. Sci Rep 2016;6:37278 47. Bonnard C, Durand A, Peyrol S, Chanseaume E, Chauvin MA, Morio B, Vidal H, Rieusset J: Mitochondrial dysfunction results from oxidative stress in the skeletal muscle of diet-induced insulin-resistant mice. J Clin Invest 2008;118:789-800 48. Feriod CN, Oliveira AG, Guerra MT, Nguyen L, Richards KM, Jurczak MJ, Ruan HB, Camporez JP, Yang X, Shulman GI, Bennett AM, Nathanson MH, Ehrlich BE:

27 Diabetes Page 28 of 91

Hepatic Inositol 1,4,5 Trisphosphate Receptor Type 1 Mediates Fatty Liver. Hepatol Commun 2017;1:23-35 49. Li H, Yu X: Emerging role of JNK in insulin resistance. Curr Diabetes Rev 2013;9:422-428 50. Sabio G, Kennedy NJ, Cavanagh-Kyros J, Jung DY, Ko HJ, Ong H, Barrett T, Kim JK, Davis RJ: Role of muscle c-Jun NH2-terminal kinase 1 in obesity-induced insulin resistance. Mol Cell Biol 2010;30:106-115

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FIGURE LEGENDS

Figure 1. PDK4 resides and interacts with IP3R1-GRP75-VDAC1 complex at MAM

interface.

(A) Immunoblot analysis of subcellular fractions isolated from mice gastrocnemius muscle.

Markers for MAM and SR/ER (protein disulfide-isomerase; PDI), mitochondria (cytochrome

c oxidase; COX IV), nucleus (proliferating cell nuclear antigen; PCNA), and cytosol (-

tubulin).

(B) In situ PLA in VXY control and PDK4-flag C2C12 myoblasts (Scale Bars, 10μm).

(C) Co-IP analysis in mice gastrocnemius muscle homogenate.

(D) Co-IP analysis in VXY (control) and PDK4-flag expressing C2C12 myotubes.

(E) Schematic illustration depicting detection of protein-protein interaction by in situ PLA at

the MAM interface using ER-targeted Sec61B-GFP and mitochondria-targeted Mito-BFP.

(F) Confocal microscope imaging of in situ PLA blobs at the MAM interface (indicated by

white arrows) in PDK4-flag expressing C2C12 myoblasts (Scale Bars, 5μm; Inset-Yellow

Scale Bars, 1 μm).

Figure 2. Inhibition of PDK4 kinase activity suppresses MAM formation.

(A–B) In situ PLA in VXY and PDK4-flag expressing C2C12 myoblasts -/+ 4mM DCA for 6

h (respective panels are quantified below). (Scale Bars, 20 μm; Mean ±SD of n=3 of more

than 30 cells/experiment; *P<0.05, **P<0.01; one-way ANOVA)

(C) Co-IP analysis in VXY and PDK4-flag expressing C2C12 myotubes -/+ 4mM DCA for

6h.

(D) Input controls of figure C.

(E) Quantification of figure C (Mean ±SD of n=3; *P<0.05, **P<0.01; Student’s t test)

29 Diabetes Page 30 of 91

(F) In situ PLA in C2C12 myoblasts were transfected with either pcDNA/ΔPDK4-flag

(respective panels are quantified below). (Scale Bars, 20μm; Mean ±SD of n=3 of more than

30 cells/experiment; ns: not significant).

Figure 3. Obesity enhances MAM formation in skeletal muscle.

(A) Immunoblot analysis of gastrocnemius muscle isolated from CD and HFD-fed mice.

(Right) Quantification (Mean ±SD of n=6/group; **P<0.01; Student’s t test)

(B) Immunoblot analysis of gastrocnemius muscle isolated from WT and ob/ob mice. (Right)

Quantification (Mean ±SD of n=3/group; **P<0.01; Student’s t test)

(C) Representative immunoblot analysis of subcellular fractions isolated from gastrocnemius muscle of CD or HFD fed mice. (Right) Quantification of MAM fractions (Mean ±SD of n=3/group; *P<0.05, **P<0.01; Student’s t test).

(D) In situ PLA in isolated myofibers from CD and HFD fed mice (respective panels are quantified below). (Scale Bars, 100μm; Mean±SD of ~10-15 myofibers/ samples; *P<0.05,

**P<0.01; Student’s t test)

(E) Immunoblot analysis of subcellular fractions isolated from gastrocnemius muscle of WT and ob/ob mice. (Below) Quantification of MAM fractions (representative of gastrocnemius skeletal muscle pooled from n=6 mice/group; Mean ±SD of n=3; *P<0.05, **P<0.01;

Student’s t test)

(F) In situ PLA in isolated myofibers from WT and ob/ob mice (respective panels are quantified below). (Scale Bars, 100μm; Mean ±SD of ~10-20 myofibers/ samples; *P<0.05,

**P<0.01; Student’s t test).

(G) Co-IP analysis in PDK4-flag overexpressing C2C12 myotubes treated with 0.4mM

Pal/BSA for 16h.

(H) Input control of figure G.

(I) Quantification of figure G (Mean ±SD of n=3; *P<0.05; Student’s t test)

30 Page 31 of 91 Diabetes

Figure 4. Genetic ablation of PDK4 reduced MAM formation in diet-induced obese

mice skeletal muscle.

(A) Immunoblot analysis of subcellular fractions isolated from CD or HFD-fed WT and

Pdk4-/- mice.

(B) Quantification of proteins in MAM fraction of figure A (gastrocnemius muscle pooled

from n=6 mice/group and 2 independent experiments were performed; *P<0.05, **P<0.01;

one-way ANOVA).

(C) In situ PLA in isolated myofibers from CD or HFD-fed WT and Pdk4-/- mice (Scale Bars,

100μm). (Right) Quantification of in situ PLA blobs (Mean ±SD of ~10-20 myofibers/

samples; *P<0.05, **P<0.01; one-way ANOVA).

(D) Representative TEM images showing the association of SR/ER and intermyofibrilar

mitochondria (IMF) in gastrocnemius muscle isolated from CD or HFD-fed WT and Pdk4-/-

mice (Red arrows indicate the SR-mitochondria contact sites) (Scale Bars, 200nm). (Inset)

Yellow dotted line indicating the surface area of MAM; Mitochondria (M); Sarcoplasmic

reticulum (SR); T-Tubule (TT) (Inset Scale Bar, 100nm).

(E) Percentage of MAM surface area per mitochondrion perimeter in each microscopic field.

(n=20/group; Mean ±SD, *P<0.05, **P<0.01; one-way ANOVA)

(F) Percentage of tight MAM (<15nM) and loose MAM (15-30nm) in total MAM surface

area (n=28~41/group; Mean ±SD, *P<0.05, **P<0.01; #P<0.05 control vs WT HFD,

&P<0.05 WT HFD vs Pdk4-/- HFD; one-way ANOVA).

Figure 5. PDK4 inhibition attenuates MAM mediated Ca2+ transfer from ER to

mitochondria, mitochondrial dysfunction and ER stress

2+ 2+ (A) Mitochondrial Ca flux was measured using 4mitD3 probe (Ai). Basal [Ca ]mito (Aii)

2+ and [Ca ]mito after stimulation with 500nM ATP (Aiii) in C2C12 myoblast treated with

31 Diabetes Page 32 of 91

50M Pal/BSA and -/+ 4mM DCA for 16 h. (n=9~12/group; Mean ±SD, *P<0.05, **P<0.01; one-way ANOVA).

2+ 2+ 2+ (B) ER lumen Ca was measured using D1ER (Bi). Basal [Ca ]ER (Bii) and [Ca ]ER after stimulation with 1µM CPA (Biii) in C2C12 myoblast treated with 50M Pal/BSA and -/+

4mM DCA for 16 h. (n=9~12/group; Mean ±SD, **P<0.01; one-way ANOVA)

2+ 2+ 2+ (C) Cytosolic Ca measurement using Fura-2 dye (Ci). Basal [Ca ]cyto (Cii) and [Ca ]cyto after stimulation with 1µM CPA (Ciii) in C2C12 myoblast treated with 50M Pal/BSA and -

/+ 4mM DCA for 16 h. (n=9~12/group Mean ±SD; one-way ANOVA).

(D) Evaluation of mitochondrial ROS generation after 16 h incubation with 0.4mM Pal/BSA in siCon/siPDK4 transfected C2C12 myotubes using CM-H2XRos normalized by

MitoTracker green FM (n=3; Mean ±SD, *P<0.05, **P<0.01; one-way ANOVA).

(E) Mitochondrial membrane potential in siCon/siPDK4 transfected C2C12 myotubes using

JC-1 dye by FACs. Graph indicates the JC-1 Red and Green ratio (n=3; Mean ±SD,

**P<0.01; one-way ANOVA).

(F) Cellular ATP level was measured in siCon/siPDK4 transfected C2C12 myotubes by

ATPlite luminescence assay. (n=3; Mean ±SD, *P<0.05, **P<0.01; one-way ANOVA).

(G) Quantitative RT-PCR measurement of mtDNA (mitochondrial ND1) normalized by gDNA (nuclear Percam1) in gastrocnemius muscle of CD or HFD-fed WT and Pdk4-/- mice

(n=5/group; Mean ±SD, **P<0.01; one-way ANOVA).

(H) Representative immunoblot analysis of ER stress markers in gastrocnemius muscle tissue of CD or HFD fed WT and Pdk4-/- mice.

(I) Quantification of figure H (Mean ±SD of n=4~5/group; *P<0.05, **P<0.01; one-way

ANOVA)

(J) Representative immunoblot analysis of ER stress markers in siCon/siPDK4 transfected

C2C12 myotubes.

32 Page 33 of 91 Diabetes

(K) Quantification of figure J (Mean ±SD of n=4; *P<0.05, **P<0.01; one-way ANOVA)

Figure 6. PDK4 deficiency attenuates palmitate-induced skeletal muscle IR via

downregulation of MAM.

(A) Immunoblot analysis in siCon/siPDK4 transfected C2C12 myotubes. (Below)

Quantification (Mean ±SD of n=3; *P<0.05, **P<0.01; one-way ANOVA)

(B) Immunoblot analysis in siCon/siPDK4 transfected C2C12 myotubes following 100 nM

insulin treatment for 10 mins. (Below) Quantification (Mean ±SD of n=3; *P<0.05; one-way

ANOVA).

(C) Immunoblot analysis in gastrocnemius muscle homogenate isolated CD or HFD fed WT

or Pdk4-/- mice. (Right) Quantification (Mean ±SD of n=3/group; *P<0.05, **P<0.01; one-

way ANOVA).

(D) Immunoblot analysis of insulin action was evaluated in CD or HFD fed WT or Pdk4-/-

mice. (Below) Quantification (Mean ±SD of n=3~4/group; *P<0.05; one-way ANOVA).

(E) Graphical representation of synthetic ER-mitochondria linker: linker-RFP HA (Linker-

RH) mediated MAM induction.

(F) Targeting of Linker-RH at MAM interface was examined in C2C12 myoblast using mito-

BFP (mitochondria) and Sec61B (ER) by confocal microscopy (Scale Bars, 20μm).

(G) Pearson’s coefficient for co-localization between mito-BFP (mitochondria) and Sec61B

(ER) in pcDNA or Linker-RH transfected C2C12 myoblast (Mean ±SD of n=8; **P<0.01;

Student’s t test).

(H) Basal and stimulated (with 500nM ATP) mitochondrial Ca2+ flux was measured in mock

or Linker-RH transduced C2C12 myoblast using 4mitD3 probe (n=9~12/group; Mean ±SD,

*P<0.05, **P<0.01; Student’s t test).

33 Diabetes Page 34 of 91

(I) Immunoblot analysis after mock or Linker-RH transduction in WT or Pdk4-/- primary myotubes. (Below) Quantification (Mean ±SD of n=3/group; *P<0.05, **P<0.01; Student’s t test)

(J) AKT activation, following 10 mins of 100nM insulin stimulation, was evaluated in Mock or Linker-RH transduced WT or Pdk4-/- primary myotubes. (Below) Quantification (Mean

±SD of n=3/group; *P<0.05, ns – no significance; Student’s t test)

(K) JNK phosphorylation was evaluated after Mock or Linker-RH transduction in DMSO or

20μM SP600125 treated for 6 h in WT primary myotubes. (Below) Quantification (Mean

±SD of n=3; *P<0.05, **P<0.01; Student’s t test)

(L) Insulin–induced AKT activation was evaluated in Mock or Linker-RH transduced WT primary myotubes treated with DMSO or 20μM SP600125 for 6 h. (Below) Quantification

(Mean ±SD of n=3; *P<0.05; Student’s t test)

Figure 7. A graphical representation demonstrating the role of PDK4 in obesity-induced

MAM formation and skeletal muscle IR.

In skeletal muscle, PDK4 interacts and stabilizes IP3R1-GRP75-VDAC1 complex at MAM interface. In physiological condition, PDK4 is expressed at low level and moderate Ca2+ transfer from ER to mitochondria via IP3R1-GRP75-VDAC1 complex formation stimulates mitochondrial ATP production. However, obesity or free fatty acid (FFA)-induced overexpression of PDK4 via Foxo1 activation augments MAM formation and promoted mitochondrial Ca2+ accumulation, mitochondrial dysfunction and ER stress. Subsequently,

ER stress-mediated JNK activation leads to inhibition of insulin signaling pathway.

34 Page 35 of 91 Diabetes

Figure 1

A B Flag-IP3R1 Flag-GRP75 PDK1 VXY PDK4-flag VXY PDK4-flag PDK2 PDK3 C

PDK4 GRP75 IP3R1 IP3R1 GRP75 Flag-VDAC1 Flag-RyR1 VDAC1 VXY PDK4-flag VXY PDK4-flag PDK4 VDAC1 RyR1 RyR1 IgG PDI COX IV β-Tubulin

D E F IP3R1-VDAC1 Inset

Flag IP3R1 flag - IP3R1-Flag GRP75

VDAC1 PDK4 IgG IP (Flag) Input Diabetes Page 36 of 91

Figure 2

A B Flag-IP3R1 Flag-GRP75 Flag-VDAC1 IP3R1-GRP75 GRP75-VDAC1 IP3R1-VDAC1

VXY VXY VXY VXY VXY VXY

PDK4-flag PDK4-flag PDK4-flag PDK4-flag PDK4-flag PDK4-flag

PDK4Flag--flag+DCAIP3R1 PDK4 Flag-flag+DCAGRP75 PDK4 Flag--flag+DCAVDAC1 PDK4IP3R1Flag--flag+DCAIP3R1-GRP75 VDAC1 PDK4 Flag-GRP75flag+DCA PDK4 IP3R1 Flag--flag+DCAVDAC1-VDAC1 Flag/IP3R1 Flag/GRP75 Flag/VDAC1 IP3R1/GRP75 GRP75/VDAC1 IP3R1/VDAC1 * ** 50 40 25 ** 1.5 5 2.0 * ** ** ** ** 40 ** 20 4 30 1.5 1.0 30 15 3 20 1.0 20 10 2 0.5 (Fold change) (Fold (Fold change) (Fold change) (Fold change) (Fold (Fold change) (Fold 10 change) (Fold 0.5 No. of Blobs/cell of No. No. of Blobs/cell of No. Blobs/cell of No. Blobs/cell of No. 10 Blobs/cell of No. 5 Blobs/cell of No. 1

0 0 0 0.0 0 0.0 Y g g Y g Y g g Y g A Y A a A a A Y a A a A X X X fl C X fl X fl C X fl C V -fla V -fla V - V - DC V - V - D + DC + DC + D + K4 + D + DK4 g DK4 g DK4 DK4 g D DK4 g P la P la P lag P la P lag P la -f f -f -f f f - - 4- DK4 DK4 DK4 DK4 DK4 DK P P P P P P

Flag IP C D Input F IP3R1-GRP75 GRP75-VDAC1 IP3R1-VDAC1 VXY PDK4-flag VXY PDK4-flag DCA - - + DCA - - +

IP3R1 IP3R1

GRP75 GRP75 pcDNA pcDNA pcDNA VDAC1 VDAC1 Flag Flag

IgG β-Actin ΔPDK4-flag ΔPDK4-flag ΔPDK4-flag 2.0 IP3R1-GRP75 VDAC1-GRP75 IP3R1-VDAC1 -DCA E ns * 1.5 1.5 1.5 +DCA ns 1.5 ns ** * 1.0 1.0 1.0 1.0

0.5 0.5 0.5 0.5 (Foldchange) (Foldchange) (Foldchange) Normalized by IgG by Normalized No. of Blobs/cell of No. No. of Blobs/cell of No. No. of Blobs/cell of No.

Desitometry (Fold change) (Fold Desitometry 0.0 0.0 0.0 0.0 1 5 A A C1 A ag N lag 3R P7 A N ag N fl f P D D -fl D D 4- I GR 4 c 4- c V pc K p K p K D D D P P P D D D Page 37 of 91 Diabetes

Figure 3

A B WT ob/ob CD HFD CD WT PDK1 HFD PDK1 ob/ob ** 2.5 4 PDK2 ** PDK2 2.0 3 1.5 PDK3 PDK3 2 1.0 PDK4 0.5 PDK4 1

normalized by HSP90 by normalized 0.0 0 GAPDH GAPDH by normalized HSP90 1 2 3 4 1 Densitometry (fold change) (fold Densitometry K K K K change) (fold Densitometry K4 D D D D DK DK2 DK3 D P P P P P P P P

C Total Mito MAM E Total Mito MAM CD HFD CD HFD CD HFD ob ob ob PDK4 * 8 CD WT ob/ WT ob/ WT ob/ IP3R1 HFD 6 * PDK4 GRP75 ** 4 * IP3R1 VDAC1 GRP75 2 PDI fraction MAM Densitometry of Densitometry VDAC1 normalized by PDI by normalized 0 COX IV 4 1 5 1 PDI K R 7 C D 3 P A P IP R D β-tubulin G V CoxIV β-tubulin D IP3R1-GRP75 GRP75-VDAC1 IP3R1-VDAC1 WT ob/ob

4 * ** 3 ** *

2

1 MAM fraction MAM Densitometry of Densitometry normalized by PDI by normalized CD HFD CD HFD CD HFD 0 4 1 5 1 K R 7 C D 3 P A P IP R D G V 3 2.5 5 ** * 2.0 * 4 2 1.5 3 Flag IP Input 1.0 G H 1 2 PDK4-Flag PDK4-Flag 0.5 1 (Fold change) (Fold (Fold change) (Fold (Fold change) (Fold 0 0.0 0 -ve BSA BSA Pal Pal No. of blobs/myofiber of No. No. of blobs/myofiber of No. No. of blobs/myofiber of No. D D D D D C F CD C IP3R1 H HF HF IP3R1 GRP75 GRP75 F IP3R1-GRP75 GRP75-VDAC1 IP3R1-VDAC1 VDAC1 VDAC1 Flag Flag IgG β-Actin

I BSA Pal * WT ob/ob WT ob/ob WT ob/ob 4 * * 3 4 ** 5 5 ** ** 2 3 4 4 1 3 3 2 2 2 IgG by Normalized 0 5 1 7 C1 1 1 change) (Fold Desitometry 3R1 P A (Fold change) (Fold (Fold change) (Fold IP D (Fold change) (Fold GR V 0 0 0 No. of blobs/myofiber of No. No. of blobs/myofiber of No. T b blobs/myofiber of No. T b T b o W o W /o W b/ b b/ o o o Diabetes Page 38 of 91

Figure 4

A C IP3R1-GRP75 Total Mito MAM CD HFD 2.5 CD HFD CD HFD CD HFD ** **

-/- -/- -/- -/- -/- -/- 2.0

1.5 WT Pdk4 WT Pdk4 WT Pdk4 WT Pdk4 WT Pdk4 WT Pdk4 IP3R1 1.0 (Fold change) (Fold 0.5

GRP75 blobs/myofiber of No. 0.0 /- /- T - T - VDAC1 -/- 4 W 4 -/- W k WT Pdk4 WT Pdk4 dk D CD P F Pd H D Mfn2 CD GRP75-VDAC1 HF COX IV CD HFD 4 PDI ** ** 3 β-tubulin

2

(Fold change) (Fold 1

B CD WT blobs/myofiber of No. 0 CD PDK4-/- /- /- - T - -/- 4 HFD WT WT Pdk4 WT Pdk4-/- WT k W k4 D d D C P Pd -/- HF D CD HFD Pdk4 HF 4 * * IP3R1-VDAC1 CD HFD 3 10 ** ** * * * * 2 * * 8 6 1

normalized by PDI by normalized 4

0 change) (Fold 2 Densitometry of MAM fraction MAM of Densitometry 1 5 2 blobs/myofiber of No. R C1 n 3 P7 A f 0 IP R D M G V -/- -/- /- /- WT Pdk4 WT Pdk4 T - T - 4 W 4 W k k d D CD P F Pd H D CD F H D CD HFD E F WT Pdk4-/- WT Pdk4-/- Tight MAM (<15nm) Loose MAM (15-30nm)

80 * *** *** 140 ** ** 60 120 100

200 nm 80

IMF mitochondria IMF 40 # 60 MAM % MAM SR SR & SR 20 40

% MAM surface area/ surface MAM % 20 SR M M TT M M perimeter Mitochondrion SR 0 Inset 0 - - TT -/ -/ /- - T T T - T -/ 4 SR SR SR 4 4 k4 W k SR SR W D W D d 100 nm 100 nm 100 nm 100 nm D W dk D dk C Pd F P TT TT C P F P D H D H C FD C FD H H Page 39 of 91 Diabetes

Figure 5

A B C i) 2+ i) 2+ i) 2+ [Ca ]mito [Ca ]ER [Ca ]cyto

) 1.5 5 2.5 BSA BSA )

BSA ) Palmitate 380 Palmitate 540 540 4 Palmitate /F Pal + DCA /F Pal + DCA /F Pal + DCA 340

490 2.0 1.0 3 490

2 1.5 0.5 1 ATP CPA CPA D1ER ratio (F 4mitD3 ratio (F ratio 4mitD3 0 1.0 Fura-2ratio (F 0.0 0 100 200 300 400 500 0 200 400 600 800 0 100 200 300 400 500 Time (Sec) Time (Sec) Time (Sec)

ii) Basal iii) ATP-Stimulated ii)Basal iii) CPA-Stimulated ii)Basal iii) CPA-Stimulated ) ) ) )

** ) 2.5 * 5 2.5 ) 2.0 0.5 1.5 540

** 380

* 380 540 540

* ** 540

/F ** ** /F /F /F /F 2.0 4 2.0 /F 0.4

490 1.5 340 490 340 490 490 1.0 1.5 3 1.5 0.3 1.0 1.0 2 1.0 0.2 0.5 0.5 1 0.5 0.5 0.1 D1ER ratio (F 0.0 D1ER ratio (F 4mitD3 ratio (F ratio 4mitD3 4mitD3 ratio (F ratio 4mitD3 0.0 0.0 Fura-2 ratio (F 0 0.0 Fura-2 ratio (F 0.0 t t t t t nt n A n n A n n nt nt A te te A o o C o o o CA o o nt A nt DC c c D c DC o ta C o ta C C D Co C l l c c i c i D A + A al + A a + A a + A m D A m + al Cl S S P l P l l + l S P a P al B a BS a a l al a B P B P P P BS P a BS P P P

D E F G H CD HFD 2.5 * ** 1.5 ** 1.6 ** ** 1.5 ** WT Pdk4-/- WT Pdk4-/- * ** ** 2.0 1.2 p-IRE1α 1.0 1.5 1.0 IRE1α 0.8 1.0 p-eIF2α 0.5 0.5 0.4 eIF2α (Fold change) (Fold

0.5 Change) (Fold mtDNA/gDNA Red CM-H2XRos Red ATP (fold change) (fold ATP

JC-1 Red/Geen Ratio Red/Geen JC-1 CHOP 0.0 0.0 0.0 0.0 /- - n n n n 4 n n 4 - T -/ o K4 o o K4 o o K4 o T 4 GAPDH C C DK4 iC D iC DK C D C DK k W k4 i PD i s P s P i i d d s i s iP i l i s iP s iP D W D P A s l A s a s s l s C P F a S A P al A a D H A P al s B S A P al FD BS P P B P C H BS BS BS

I J BSA Pal K

CD WT HFD WT BSA siCon Pal siCon -/- -/- BSA siPDK4 Pal siPDK4 siPDK4 siPDK4 CD Pdk4 HFD Pdk4 siCon siCon p-IRE1α 10 10 IRE1α ** * * *** * 8 p-PERK 8 p=0.06 ** ** PERK 6 ** 6 * p-eIF2α *** 4 4 * * ** * eIF2α Desitometry Densitometry (Fold Change) (Fold change) 2 CHOP 2 0 β-Tubulin 0 p-IRE11 α p-eIF22 α CHOP3 p-IRE11 α p-PERK2 p-eIF23 α CHOP4 /IRE1α /eIF2α /GAPDH /IRE1α /PERK /eIF2α /β-Tubulin Diabetes Page 40 of 91

Figure 6

BSA Pal BSA Pal BSA Pal CD HFD CD HFD -/- -/- A B D -/- -/- WT Pdk4 WT Pdk4 WT WT Pdk4 Pdk4 siPDK4 siPDK4 siCon siCon siPDK4 siPDK4 siPDK4 siPDK4 siCon siCon siCon siCon PDK4 p-AKT PDK4 AKT p-JNK1 p-IRS1s307 β-Tubulin JNK1 IRS1 β-Tubulin Insulin ---- + + + + p-AKT * 15 2.5 ** ** BSA siCon * * AKT 2.0 BSA siPDK4 10 1.5 Pal siCon Pal siPDK4 HSP90 1.0 Insulin ---- + + + + + + + + + 0.5 5 p-AKT/AKT p-JNK1/JNK1 (Foldchange) 0.0 (Fold change) CD WT n n 4 * K4 0 -/- iCo D iCo CD Pdk4 iP iPDK Insulin ----+ + + + 50 A s s s -/- Pal sal 40 HFD Pdk4 BS P 30 BSA 5 ** * HFD WT CD HFD * C 4 20 WT Pdk4-/- WT Pdk4-/- 3 * * p-JNK1 2 10 Densitometry (Fold change) p-JNK1/JNK1 JNK1 (Foldchange) 1 0 0 Insulin -+- +-+-+-+-+-+-+ /- - GAPDH - T -/ p-IRS1s307/IRS1 p-AKT/AKT k4 W k4 D WTd D d C P F P D H C FD H

E F Mito-BFP Linker-RH Sec61B Merged Inset G H 2+ [Ca ]mito ) 0.8 1.8 Mock ** 540

/F Linker-RH 1.6 * 0.6 490 * *

pcDNA ** 1.4 0.4 1.2 * 0.2

RH 1.0 - ATP 4mitD3 ratio (F ratio 4mitD3

Pearson's coefficient Pearson's 0.8 0.0 0 100 200 300 400 500 A H Linker N R Time (Sec) D r- c e p k in L

WT Pdk4-/- -/- I J WT Pdk4 K L RH RH - - RH RH - - Mock Mock SP600125 -- + - - + Linker Linker SP600125 - - + Mock Mock Mock Linker PDK4 Linker p-AKT p-AKT p-JNK1 p-JNK1 AKT AKT JNK1 JNK1 GAPDH HSP90 HSP90 HSP90 Insulin - + + + + Insulin --- + + +

ns 2.0 25 2.5 * ** * ** WT 5 * * Mock ** Mock -/- WT Mock 2.0 Pdk4 20 4 WT Linker-RH 1.5 Linker-RH Linker-RH -/- Pdk4 Mock 15 1.5 3 Pdk4-/- Linker-RH 1.0 1.0 2 10 p-AKT/AKT p-AKT/AKT p-JNK1/JNK1 p-JNK1/JNK1 (Fold change) (Fold change)

(Fold change) (Fold 0.5 0.5 (Foldchange) 1 5

0.0 0 0.0 0 Mock + +- - Insulin - + + + + SP600125 +-- SP600125 ---- + + Linker-RH - -+ + Insulin - -+ + - + Page 41 of 91 Diabetes

Figure 7 Diabetes Page 42 of 91

Supplementary Figure S1

A B C Neg control IP3R1-GRP75 GRP75-VDAC1

IP3R1

GRP75

VDAC1 GRP75

PDK4 IP3R1 IP3R1-VDAC1 GRP75-RyR1 VDAC1-RyR1 PDI VDAC1 RyR1 CoxIV IgG PCNA

β-Tubulin

D F

IP3R1 GRP75 β-Tubulin β-Tubulin

E G IP3R1-GRP75 GRP75-VDAC1 IP3R1-VDAC1 IP3R1-GRP75 GRP75-VDAC1 IP3R1-VDAC1

siCon siCon siCon siCon siCon siCon

siIP3R1 siIP3R1 siIP3R1 siGRP75 siGRP75 siGRP75

1.5 1.5 1.5 1.5 1.5 1.5

1.0 1.0 1.0 1.0 1.0 1.0 ** * ** 0.5 0.5 0.5 ** 0.5 0.5 ** 0.5 ** 0.0 0.0 No. Blobs/cell(Fold Change) Blobs/cell(Fold No. 0.0 0.0 0.0 No. Blobs/cell (Fold Change) (Fold Blobs/cell No. 0.0 No. Blobs/cell (Fold Change) (Fold Blobs/cell No. n 1 1 change) (Fold Blobs/cell of No. n o n 1 o R R change) (Fold Blobs/cell of No. 3 R n change) (Fold Blobs/cell of No. n n 5 iC 3 iC Co o 75 o 75 o 7 s IP s IP i P3 C P C P i i s iI i i RP iC s s s s s s R iGR iG iG s s s Page 43 of 91 Diabetes

Supplementary Figure S1 (Continued)

H DAPI Mito-GFP PLA blob Merged Inset Flag-IP3R1 VXY

Flag-IP3R1 flag - PDK4 Flag-GRP75

Flag-VDAC1 Diabetes Page 44 of 91

Supplementary Figure S2

PDHE1α – PDK2 A ΔPDK4-flag B IP3R1 - PDHE1α PDHE1α - VDAC1 siCon siPDHE1α 5 1.25 2.5 5 ug +ve -ve p-PDH s300

p-PDH s293

p-PDH s232

PDHE1α COX IV

80Kda D 65Kda IP3R1-GRP75 GRP75-VDAC1 IP3R1-VDAC1 50Kda

ΔPDK4-Flag 40Kda C 30Kda 25Kda PDHE1α 15Kda IB: αFlag siCon siCon siCon 10Kda β-Tubulin

E VXY

siPDHE1α siPDHE1α siPDHE1α

2.0 ns ns 1.5 ns 1.5

siCon siPDHE1α 1.5 1.0 1.0

VDAC1 PDK4 - siPDHe1α 1.0

0.5 0.5 IP3R1 (Fold change) (Fold (Fold change) (Fold (Fold Change) (Fold 0.5 No. of Blobs/cell of No. No. of Blobs/cell of No. No. of Blobs/cell of No.

0.0 0.0 0.0 n a n a n a 1 o 1 o 1 e C iC e iCo i He s H s s D D siCon DH P iP siPDHE1α iP i s s s

IP3R1-VDAC1 2.5 ** ns

2.0

1.5

1.0

0.5 (Foldchange) No. of Blobs/cell of No. 0.0 n a n a o C e1 iCo He1 H s D D P 4 si P XY i K V s D Y P 4 si X K V D P Page 45 of 91 Diabetes

Supplementary Figure S3

IP3R1-VDAC1 A B D BSA 2h BSA 4h BSA 8h BSA 16h Pal BSA BSA 1.5 PDK1 Pal * PDK2 1.0 PDK3 PDK4 0.5 Pal 2h Pal 4h Pal 8h Pal 16h GAPDH Densitometry (AU) Densitometry 0.0 normalized by GAPDH by normalized

K1 D DK2 DK3 DK4 P P P P

C Total Mito MAM E IP3R1-VDAC1 BSA Pal BSA Pal BSA Pal

PDK4 12 * BSA 6 BSA IP3R1 8 Pal Pal ** 4 GRP75 * * ** Mfn2 4 * 2 * *

VDAC1 fraction MAM (Fold change) (Fold Densitometry of Densitometry normalized by PDI by normalized

PDI 0 count/nucleus Blobs 0 5 2 h h h COX IV R1 7 n 2 4 8 6h DK4 3 1 P IP Mf DAC1 β-Tubulin GRP V

F IP3R1-VDAC1 G Pal (mM) 0 0.1 0.2 0.4 5.5mM Glu 25mM Glu 15 Glu ** *** 10 * 5.5mM 5.5mM **

5 * (Fold change) (Fold No. of blobs/nucleus of No. Glu 0

0 .1 .2 .4 0 .1 .2 .4 0 0 0 0 0 0 25mM 25mM Pal (mM)

H BSA Pal I J BSA Pal 0 2 4 8 16 2 4 8 16 h CD HFD CD PPARα PPARα 2.0 HFD PPARγ

PPARγ siCOn siCOn 1.5 * siFoxo1 siFoxo1 PGC1α PGC1α PDK4 p-FoxO1 1.0 p-FoxO1 PDK2 FoxO1 FoxO1 0.5 FoxO1 normalized by normalized β-Tubulin β-Tubulin PDK4 FoxO1 Total -Tubulin/ 0.0 b a g a β-Tubulin R 1

Densitometry (Fold Change) (Fold Densitometry R O1 A C x PA P G o P P P -F p Diabetes Page 46 of 91

Supplementary Figure S4

A CD HFD B WT Pdk4-/- WT Pdk4-/- PDK4 1.5 ** ns ns ns ns ns CD WT CD Pdk4-/-

IP3R1 -Tubulin HFD WT b 1.0 GRP75 HFD Pdk4-/- VDAC1 0.5

Mfn2 Densitometry (AU) Densitometry 0.0 RyR1 by normalized 4 5 2 1 K R1 C1 R D 3 P7 A fn y β-Tubulin P IP D M R GR V

C IP3R1-GRP75 D IP3R1-VDAC1 BSA siCon BSA siPDK4 BSA siCon BSA siPDK4 2.5 ** * 2.5 * ** ** 2.0 2.0

1.5 1.5

1.0 1.0 Pal siCon Pal siPDK4 Pal siCon Pal siPDK4 (Fold change) (Fold (Fold change) (Fold 0.5 0.5 Blobs count/nucleus Blobs Blobs count/nucleus Blobs 0.0 0.0 n 4 n 4 n 4 n 4 K K o K iCo iCo DK iCo iC D s iPD s iP s iPD s iP A s al s A s al s S A P al S A P al B S P B S P B B

CD Crude Mito BSA E F WT Pdk4-/- G -/- BSA Pal WT Pdk4 ER ER M M ER

siCon siPDK4 siCon siPDK4 M PDK4 M ER IP3R1 ER M GRP75 VDAC1 HFD Pal WT Pdk4-/- WT Pdk4-/- COX IV ER ER SS mitochondria 8 ** myoblast Primary ER * * ER M 6 ER M

4 ER

IP3R1/COX IV IP3R1/COX 2

H 1.0 I 100 0 * * 50 *** *** 0.8 n 4 4 50 o on K C DK iC D i iP s iP 0.6 40 s s l s A a l S A P a 0.4 30 B S P B 20 0.2 10 0.0

% MAM surface area/ surface MAM % 0 No. ER-linked Mito/ ER-linked No. -

total no. of Mito (AU) Mito of no. total - -/ -/ /- /- T T perimeter Mitochondrion T - T - W W 4 k4 k4 W k W k4 A al d l d S Pd P Pd A P a P B A al S P l S P B A a B S P B Page 47 of 91 Diabetes

Supplementary Figure S4 (Continued)

J WT Pdk4-/- WT WT Mfn1 -/- Pdk4-/- Pdk4 OPA1 2.0 1.5

p-Drp1 1.5 s637 1.0 p-Drp1s616 1.0 Drp1 0.5 0.5 Fis1 0.0 0.0 normalized by HSP90 by normalized 6 HSP90 1 DRP1 Total by normalized 37 1

Densitometry (Fold Change) (Fold Densitometry 6 6 Densitometry (Fold Change) (Fold Densitometry n1 A1 rp is1 s s Mf D F 1 1 OP rp rp -D -D p p siPDK4 K siCon siCon siCon Mfn1 siPDK4 siPDK4 1.5 1.5 Mfn2 -Tubulin

OPA1 b 1.0 1.0

p-Drp1s637 0.5 0.5

p-Drp1s616 0.0 0.0

Drp1 by normalized 2 1 1 6 normalized by Total DRP1 Total by normalized 37 1 Densitometry (Fold Change) (Fold Densitometry n1 n A p1 s f i Change) (Fold Densitometry 6 6 r F s s Mf M OP D 1 1 Fis1 rp rp -D -D p p β-Tubulin

WT Pdk4-/- L WT Pdk4-/- 4 4

3 3

2 2

1 1

0 0 Mitochondrial perimeter (um) perimeter Mitochondrial /- T - S

Total Mitochondrial perimeter (um) perimeter Mitochondrial Total 4 S W k IMF d P M N -/- WT Pdk4 WT Pdk4-/- 150 150

100 100

50 50

0 0 OCR (pmol/min/Norm. Unit) (pmol/min/Norm. OCR OCR (pmol/min/Norm. Unit) (pmol/min/Norm. OCR 0 20 40 60 80 100 l l re sa ima a Time (minutes) a x p B a S M Diabetes Page 48 of 91

Supplementary Figure S5

A B C CD HFD * ** -/- -/- * * WT Pdk4 WT Pdk4 ** ** * * 2.5 1.5 * * Complex V * * 2.0 Complex III 1.0 1.5 Complex IV

1.0 ATP 0.5 Complex II (Foldchange)

(Foldchange) 0.5 Red CM-H2XRos Red Complex I 0.0 0.0 1 n 5 n 5 1 n 5 1 on 75 R o R1 R C C 3 iCo iCo P7 Gapdh i i P s RP7 P3 s R s iIP3 s iI G G s l s i i siGRP s s l siIP3R Pa l siGRP7l SA A siI Pal l a BSA A a B A S P S P Pa B Pa B BSA BS

BSA Pal D CD WT HFD WT E BSA siCon Pal siCon CD Pdk4-/- HFD Pdk4-/- BSA siIP3R1 Pal siIP3R1

siCOn siIP3R1 siCOn siIP3R1 * * ** * 8 2.5 ns ns ns ns ns IP3R1 4 2.0 GRP78 4 1.5 p-PERK 3 * * 2 1.0 PERK Densitometry (Fold change) (Fold 1 0.5 CHOP 0

normalized byGAPDH 0.0 COX IV GRP78 p-PERK CHOP /COX IV /PERK /COX IV Densitometry (Fold Change) V III IV II I

F BSA Pal G DMSO Tg siCon siCon siPDK4 BSA siCon Pal siCon 0 2 4 6 2 4 6 2 4 6 h BSA siGRP75 Pal siGRP75 p-IRE1α siCOn siCOn siGRP75 siGRP75 IRE1α GRP75 6 * ** p-IRE1α ** p-JNK1 4 * * IRE1α JNK1 * * p-eIF2α p-eIF2α 2 eIF2α eIF2α Densitometry (Fold change) (Fold CHOP 0 CHOP β-Tubulin p-IREα p-eIF2α CHOP β-Actin /IREα /eIF2α /β-Tubulin

siCon DMSO H siCon DMSO I J siCon DMSO K siCon DMSO siCon Tg siCon Tg siCon Tg siCon Tg siPDK4 Tg siPDK4 Tg siPDK4 Tg siPDK4 Tg * 8 10 6 10 * * * ** 8 a * * * * 8 a * * 6 6 ** * ** * ** * 4 /eIF2 6 -Actin /IRE1 b a a 4 4 4 2 2 2 ** p-JNK1/JNK1 Chop/ (Fold change) (Fold

2 p-eIF2 (Fold Change) (Fold (Fold Change) (Fold (Fold Change) (Fold p-IRE1 0 0 0 0

h h h h h h h h h h h 0 2 4 h 6 h 0 2 h 4 h 6 h 0 2 4 6 0 2 4 6 Page 49 of 91 Diabetes

Supplementary Figure S5 (Continued)

IP3R1-VDAC1 L M DMSO Tg siCon siCon Tg siPDK4 Tg ** ** 5 Ad PDK4 ----+ + + + 4 Ad Con + +---- + + 3 siGRP75 - + - + - + - + 2 siCon + - + - + - + -

(Fold change) (Fold 1 GRP75

Blob count/nucleus Blob 0 PDK4 n g o T Tg iC n 4 s o K CHOP iC D s iP s COX IV N In presence of PDK4 Suppression of PDK4 1.5 * * ER Tg ER Tg ** ER stress ER stress 1.0

IP3R1 IP3R1 SERCA SERCA 0.5

GRP75 IV Chop/COX ? GRP75 VDAC1 Feedback 0.0 Ca2+ VDAC1 SiCon + - +- + - + - Ca2+ SiGRP75 - + - + - + - + Mito ROS Ad Con + +- - + + - - Mito ROS Ad PDK4 - - + + - - + + Diabetes Page 50 of 91

Supplementary Figure S6

BSA Pal A B BSA Pal BSA Pal C BSA Pal siCon siIP3R1 siCon siIP3R1 siCon siIP3R1 siCon siIP3R1 siCon siIP3R1 siCon siIP3R1 siCon siGRP75 siCon siGRP75 IP3R1 p-AKT GRP75

p-JNK1 AKT p-JNK1 JNK1 JNK1 β-Tubulin β-Tubulin COX IV Insulin ---- + + + + ** * 4 ** ** 5 BSA siCon 4 * * 4 3 BSA siIP3R1 3 3 Pal siCon 2 2 2 Pal siIP3R1 1 1 p-AKT/AKT 1 p-JNK1/JNK1 (Fold change) (Fold p-JNK1/JNK1 (Fold change) (Fold (Foldchange) 0 0 0 Insulin - + - + - + - + l l n 5 n 5 A A a a o 7 o 7 S S P P C P C P B B 1 i R i R n s s n 1 o R iG l iG o R iC 3 A s a s iC 3 s IP S P l s IP i B A a i s S P s B

D BSA Pal BSA Pal E F siCon siGRP75 siCon siGRP75 siGRP75 siGRP75 siCon siCon HA p-AKT HA p-AKT AKT p-JNK1 AKT β-Actin JNK1 β-Tubulin Insulin ---- + + + + 4 Insulin --- + + + ** ** ** 3 30 BSA siCon 5 ** Mock 20 BSA siGRP75 2 4 Linker RH Pal siCon 10 1 3 p-JNK1/JNK1 (Fold change) (Fold Pal siGRP75 2 2 0 p-AKT/AKT p-AKT/AKT

(Fold change) (Fold 1 (Fold change) (Fold k 1 c H o r-R M e 0 k 0 in Insulin - + - + - + - + L Insulin - + - +

G H

siPDK4 -- + - - + siPDK4 - - + ** siCon + + - + + - 8 Mock siCon siCon + + - 6 * ** * Linker-RH siCon PDK4 p-AKT 6 4 Linker-RH siPDK4 p-JNK1 4 AKT 2 JNK1 p-AKT/AKT 2 (Fold change) (Fold p-JNK1/JNK1 (Fold change) (Fold β-Actin HSP90 0 0 Insulin --- + + + Insulin - + - + - + on Con DK4 si siC iP k H s R H - R Moc er - k er in k L in L Page 51 of 91 Diabetes

Supplementary Table S1

Antibody Name Provider Catalog No. Application Dilution IP3R1 Santa Cruz sc-28614 IB/in situ PLA 1:1000/1:50 GRP75 Santa Cruz sc-133137 IB/in situ PLA/IP 1:1000/1:100/1:100 PERK Santa Cruz sc-13073 IB 1:1000 PDK2 Santa Cruz sc-100534 IB/in situ PLA 1:1000/1:50 PGC1α Santa Cruz sc-5816 IB 1:1000 GRP78 Santa Cruz sc-1050 IB 1:1000 eIF2α Santa Cruz sc-11386 IB 1:1000 Fis1 Santa Cruz sc-98900 IB 1:1000 VDAC1 Santa Cruz sc-390996 IB/in situ PLA 1:1000/1:100 Drp1 Santa Cruz sc-271583 IB 1:1000 VDAC1 Abcam ab14734 IB/in situ PLA 1:1000/1:100 COX IV Abcam ab16056 IB 1:1000 IP3R1 Abcam ab166871 IB/in situ PLA 1:1000/1:100 PPARα Abcam ab2779 IB 1:1000 p-IRE1α Abcam ab4817 IB 1:1000 Mfn1 Abcam ab57602 IB 1:1000 Total Oxphos-Complex Abcam ab110413 IB 1:1000 PDI Abcam ab2792 IB 1:1000 Ryr1 Cell signaling #8153 IB 1:1000 PDK1 Cell signaling #3820 IB 1:1000 Flag Cell signaling #2368 IB/in situ PLA 1:1000/1:100 PDHE1α Cell signaling #3205 IB/in situ PLA 1:1000/1:100 Mfn2 Cell signaling #9482 IB 1:1000 p-Drp1 s616 Cell signaling #3455 IB 1:1000 p-Drp1 s637 Cell signaling #4867 IB 1:1000 PPARγ Cell signaling #2435 IB 1:1000 p-Foxo1 Cell signaling #9461 IB 1:1000 Foxo1 Cell signaling #2880 IB 1:1000 p-PERK Cell signaling #3179 IB 1:1000 IRE1α Cell signaling #3294 IB 1:1000 p-eIF2α Cell signaling #9721 IB 1:1000 p-JNK Cell signaling #9251 IB 1:1000 Chop Cell signaling #2895 IB 1:1000 JNK Cell signaling #9252 IB 1:1000 p-AKT Cell signaling #9271 IB 1:1000 AKT Cell signaling #2920 IB 1:1000 HSP90 Cell signaling #4874 IB 1:1000 GAPDH Cell signaling #2118 IB 1:1000 HA Cell signaling #2367 IB 1:1000 p-PDH s232 Calbiochem AP1063 IB 1:10000 p-PDH s293 Calbiochem AP1062 IB 1:10000 p-PDH s300 Calbiochem AP1064 IB 1:10000 β-tubulin ABM AMB-G098 IB 1:1000 β-Actin Sigma A5441 IB 1:2000 Flag Sigma F1804 IB/in situ PLA 1:10000/1:500 p-IRS-1 Upstate 07-247 IB 1:1000 IRS-1 Upstate 06-248 IB 1:1000 PCNA BD biosciences #610664 IB 1:1000 OPA1 BD biosciences #612606 IB 1:1000 PDK3 Anti-serum NA IB 1:1000 PDK4 Anti-serum NA IB 1:1000 Diabetes Page 52 of 91

SUPPLEMENTAL MATERIALS

FIGURE LEGENDS

Figure S1. IP3R1, GRP75 and VDAC1 form a complex and interact with PDK4 in

MAM interface, Related to Figure 1.

(A) Immunoblot of subcellular fractions isolated from C2C12 myotubes.

(B) Validation of interaction between MAM proteins IP3R1, GRP75 and VDAC1 by in situ

PLA in C2C12 myoblasts (Scale Bars, 20μm).

(C) Co-IP with GRP75 antibody in C2C12 myotubes.

(D) & (F) Validation of IP3R1 and GRP75 knockdown in C2C12 myoblast.

(E) & (G) Evaluation of IP3R1-GRP75-VDAC1 interaction after knocking down IP3R1 or

GRP75 by in situ PLA in C2C12 myoblast (Scale Bars, 20μm). Following bar graph represents the quantification plot of in situ PLA blobs of each respective panel. (Mean ±SD of n=3 of more than 30 cells per experiment; *P<0.05, **P<0.01; Student’s t test)

(H) Evaluation of interaction site of PDK4 with MAM proteins by aligning the in situ PLA blobs with mito-GFP using confocal microscope (White Scale Bars, 10μm; Inset-Yellow

Scale Bars, 2.5μm).

Figure S2. Regulation of MAM integrity by PDK4 is PDH independent, Related to

Figure 2.

(A) Dose dependent overexpression ΔPDK4-flag and evaluation of PDH phosphorylation in

C2C12 myoblast by immunoblot.

(B) Evaluation of interaction between PDHE1α and IP3R1/VDAC1 by in situ PLA in C2C12 myoblast. Interaction between PDHE1α and PDK2 in absence or presence of siPDHE1α was used as negative (-ve) and positive (+ve) control respectively (Scale Bars, 20μm).

(C) Validation of PDHE1α knockdown by immunoblot.

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Page 53 of 91 Diabetes

(D) Evaluation of IP3R1-GRP75-VDAC1 interaction in PDHE1α knockdown C2C12

myoblast by in situ PLA (Scale Bars, 20μm). Following bar graph represents the

quantification plot of in situ PLA blobs of each respective panel. (Mean ±SD of over 30 cells

for each condition; ns –no significance; Student’s t test).

(E) Evaluation of IP3R1 and VDAC1 interaction in VXY or PDK4-flag expressing C2C12

myoyubes in absence or presence of siPDHE1α by in situ PLA (Scale Bars, 20μm). (Below)

Quantification (Mean ±SD of over 30 cells for each condition; **P<0.01; ns – no

significance; one-way ANOVA)

Figure S3. Saturated fatty acid palmitate enhanced MAM formation in skeletal muscle

cells, Related to Figure 3.

(A) PDKs protein expression level after 0.4mM Pal/BSA treatment for 16 h in C2C12

myotubes.

(B) Quantification of figure A (Mean ±SD of n=3; *P<0.05; Student’s t test)

(C) Immunoblot of subcellular fractions isolated from C2C12 myotubes treated with 0.4mM

Pal/BSA for 16 h. (Right) Quantification of MAM fractions (Mean ±SD of n=3; *P<0.05,

**P<0.01; Student’s t test)

(D) in situ PLA of IP3R1-VDAC1 interaction in C2C12 myotubes treated with 0.4mM

Pal/BSA at various time points (hour – h) (Scale Bars, 20μm).

(E) Quantification of in situ PLA blobs of figure D. (Mean ±SD of over 30 cells for each

condition; *P<0.05, **P< 0.01; Student’s t test)

(F) IP3R1 and VDAC1 interaction was monitored by in situ PLA after treatment of

increasing dose of Pal for 16 h (as indicated in the figure) with low glucose (5.5mM) or high

glucose (25mM) media (as indicated in the figure) in C2C12 myotubes (Scale Bars, 20μm).

(G) Quantification of in situ PLA blobs of figure F. (Mean ±SD of over 30 cells for each

condition; *P<0.05, **P<0.01, ***P<0.001; one-way ANOVA)

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Diabetes Page 54 of 91

(H) Transcription factors of PDK4 was evaluated by Immunoblot after incubating with

0.4mM Pal/BSA at various time points in C2C12 myotubes.

(I) Immunoblot of gastrocnemius muscle homogenate isolated from 16 weeks CD and HFD fed mice. (Right) Quantification (Mean ±SD of n=2/group; *P<0.05; Student’s t test)

(J) Examination of PDK4 and PDK2 protein expression level after knocking down FOXO1 by immunoblot.

Figure S4. PDK4 deficiency reduced high fat diet or palmitate-induced MAM formation without affecting the expression levels of MAM and mitochondrial dynamics related proteins, Related to Figure 4.

(A) Evaluation of total protein expression levels of indicated MAM resident proteins in gastrocnemius muscle isolated from CD or HFD fed WT or Pdk4-/- mice by immunoblot.

(B) Quantification of figure A (Mean ±SD of n=3; **P<0.01, ns – no significance; one-way

ANOVA)

(C) & (D) Evaluation of interaction between IP3R1 and GRP75/VDAC1 by in situ PLA in siCon/siPDK4 transfected C2C12 myotubes after incubating with 0.4mM Pal/BSA for 16 h.

(Right) Quantification of in situ PLA blobs of each respective panel (Scale Bars, 10μm)

(Mean ±SD of n=3 of over 30 cells per experiment; *P<0.05, **P<0.01; one-way ANOVA).

(E) Examination of IP3R1 enrichment in crude mitochondria isolated from siCon/siPDK4 transfected C2C12 myotubes after incubating with 0.4mM Pal/BSA for 16 h. (Below)

Quantification (Mean ±SD of n=3; *P<0.05, **P<0.01; one-way ANOVA).

(F) Representative TEM images of subsarcolemmal (SS) mitochondria in gastrocnemius muscle isolated from CD or HFD-fed WT and Pdk4-/- mice.

(G) Representative TEM images of ER-mitochondria contacts in primary myoblast isolated from WT or Pdk4-/- mice after stimulating 0.4mM Pal/BSA for 16 h. (Scale Bars, 500nm).

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Page 55 of 91 Diabetes

(H) Number of mitochondria linked with ER in total number of mitochondria in each

microscopic field (n=10/group; Mean ±SD, *P<0.05, **P< 0.01; one-way ANOVA).

(I) Percentage of MAM surface area per mitochondrion perimeter in each microscopic field

(n=20/group; Mean ±SD, ***P< 0.001; one-way ANOVA).

(J) Evaluation of mitochondrial dynamics-related proteins in WT and Pdk4-/- mice. (Right)

Quantification (Mean ±SD of n=3/group; Student’s t test).

(K) Evaluation of mitochondrial dynamics related proteins in C2C12 myotubes transfected

with siCon/siPDK4. (Right) Quantification (Mean ±SD of n=3; Student’s t test).

(L) TEM images showing the mitochondria morphology in gastrocnemius muscle isolated

from WT and Pdk4-/- mice (Scale Bars, 2um). (Right) Total mitochondria perimeter (um) and

mitochondrial perimeter of SS and IMF (um) (n=74~123).

(M) OCR level was measured in primary myotubes isolated from WT/ Pdk4-/- mice

gastrocnemius.

(N) Quantification of Figure M.

Figure S5. Attenuation of MAM formation prevented palmitate-induced mitochondrial

dysfunction, ER stress and insulin resistance, Related to Figure 5.

(A) Mitochondrial ROS generation measurement in siCon/siIP3R1si/GRP75 transfected

C2C12 myotubes after incubating with 0.4mM Pal/BSA for 16 h using CM-H2XRos

normalized by MitoTracker green FM (Mean ±SD of n=2, *P<0.05, **P< 0.01; one-way

ANOVA).

(B) Cellular ATP was measured in siCon/siIP3R1/siGRP75 transfected C2C12 myotubes

after incubating with 0.4mM Pal/BSA for 16 h by ATPlite luminescence assay. (n=3; Mean

±SD, *P<0.05, **P<0.01; one-way ANOVA).

(C) Immunoblot of mitochondrial total OXPHOS-complex proteins in gastrocnemius muscle

isolated from 16weeks CD or HFD-fed WT and Pdk4-/- mice.

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Diabetes Page 56 of 91

(D) Quantification of figure C (Mean ±SD of n=6/group; ns – no significance; one-way

ANOVA)

(E-F) Palmitate induced ER stress was analyzed by immunoblot in IP3R1 or GRP75 knockdown C2C12 myotubes. (Right) Quantifications (Mean ±SD of n=3, *P<0.05,

**P<0.01; one-way ANOVA).

(G) siCon/siPDK4 transfected C2C12 myotubes were incubated with 0.25μM Tg

(thapsigargin) at different time points (hour – h) and examined the ER stress markers by immunoblot.

(H-K) Quantification of figure G (Mean ±SD of n=2, *P<0.05, **P<0.01; one-way

ANOVA).

(L) IP3R1 and VDAC1 interaction were examined by in situ PLA in siCon/siPDK4 transfected C2C12 myotubes after incubating with 0.25μM Tg for 6 h (Scale Bars, 20μm).

(Right) in situ PLA blobs quantification (Mean ±SD of more than 30 cells per condition;

**P<0.01; one-way ANOVA).

(M) C2C12 myotubes were transfected or transduced with siGPR75 or adeno virus (Ad)

PDK4 or both siGRP75 and adPDK4 followed by treatment of 0.25μM Tg for 6 h. ER stress marker, CHOP expression was evaluated by immunoblot. (Below) Quantification (Mean ±SD of n=2, *P<0.05, **P<0.01; one-way ANOVA).

(N) Schematic representation, showing that PDK4 enhances Tg-induce ER stress through induction of MAM formation, possibly via a feedback mechanism induced by enhanced generation of mitochondria ROS. Whereas, suppression of PDK4 reduces Tg-induced ER stress by attenuating MAM formation

Figure S6. MAM suppression protects from palmitate-induced insulin resistance,

Related to Figure 6.

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(A) & (C) JNK phosphorylation was analyzed in siCon/siIP3R1/siGRP75 transfected C2C12

myotubes after incubating with 0.4mM BSA/Pal for 16 h by immunoblot. (Below)

Quantification of each respective figures (Mean ±SD of n=3; *P<0.05, **P<0.01; one-way

ANOVA)

(B) & (D) Insulin-stimulated AKT phosphorylation was evaluated in siIP3R1/siGRP75

transfected C2C12 myotubes after incubating with 0.4mM BSA/Pal for 16 h (Below)

Quantification of each respective figures (Mean ±SD of n=3; *P<0.05, **P<0.01; one-way

ANOVA)

(E) Mock or Linker-RH was transduced in C2C12 myotubes and JNK phosphorylation was

examine by immunoblot. (Below) Quantification (Mean ±SD of n=3; **P<0.01; Student’s t

test)

(F) AKT phosphorylation was evaluated by immunoblot in mock or Linker-RH

overexpressing C2C12 myotubes. (Below) Quantification (Mean ±SD of n=3; **P<0.01;

Student’s t test)

(G) Immunoblot analysis after mock or Linker-RH transduction in control or PDK4

knockdown C2C12 myotubes. (Right) Quantification (Mean ±SD of n=3; *P<0.05; one-way

ANOVA)

(H) Insulin-stimulated AKT phosphorylation was evaluated after mock or Linker-RH

transduction in control or PDK4 knockdown C2C12 myotubes. (Right) Quantification (Mean

±SD of n=3; **P<0.01; one-way ANOVA)

Table S1. Antibody information

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Diabetes Page 58 of 91

PDK4 augments ER-mitochondria contact to dampen skeletal muscle insulin signaling during obesity (#DB18-0363)

Full unedited blots and microscope images for supplementary figures Page 59 of 91 Diabetes

Full unedited blots and microscope images for supplementary figures

Figure S1A

IP3R1

GRP75

VDAC1

PDK4

PDI

COX IV

PCNA

β-tubulin Diabetes Page 60 of 91

Full unedited blots and microscope images for supplementary figures

Figure S1B

Neg control IP3R1-GRP75 GRP75-VDAC1

IP3R1-VDAC1 GRP75-RyR1 VDAC1-RyR1 Page 61 of 91 Diabetes

Full unedited blots and microscope images for supplementary figures

Figure S1C

GRP75

IP3R1

VDAC1

RyR1

IgG Diabetes Page 62 of 91

Full unedited blots and microscope images for supplementary figures

Figure S1D & S1F Figure S1D

IP3R1

β-tubulin

Figure S1F

GRP75

β-tubulin Page 63 of 91 Diabetes

Full unedited blots and microscope images for supplementary figures

Figure S1E

siCon siIP3R1 IP3R1-GRP75

siCon siIP3R1 GRP75-VDAC1

siCon siIP3R1 IP3R1-VDAC1 FigureS1G

IP3R1-VDAC1 GRP75-VDAC1 IP3R1-GRP75 Full unedited blots microscope and images forsupplementary figures siCon siCon siCon Diabetes siGRP75 siGRP75 siGRP75 Page 64of91 Page 65 of 91 Diabetes

Full unedited blots and microscope images for supplementary figures

Figure S1H Diabetes Page 66 of 91

Full unedited blots and microscope images for supplementary figures

Figure S2A

pPDH s300

pPDH s293

pPDH s232

PDHe1α

ΔPDK4-Flag Page 67 of 91 Diabetes

Full unedited blots and microscope images for supplementary figures

Figure S2B

PDHE1α-PDK2

IP3R1-PDHE1α PDHE1α-VDAC1 siCon siPDHE1α

Figure S2C

PDHE1α

Β-tubulin Diabetes Page 68 of 91

Full unedited blots and microscope images for supplementary figures

Figure S2D

IP3R1-GRP75 GRP75-VDAC1 IP3R1-VDAC1 siCon siPDHE1α

Figure S2E VXY

PDK4 IP3R1-VDAC1 Page 69 of 91 Diabetes

Full unedited blots and microscope images for supplementary figures

Figure S3A

PDK1

PDK2

PDK3

PDK4

GAPDH Diabetes Page 70 of 91

Full unedited blots and microscope images for supplementary figures

Figure S3C

PDK4

IP3R1

GRP75

Mfn2

VDAC1

PDI

COX IV

β-Tubulin Page 71 of 91 Diabetes

Full unedited blots and microscope images for supplementary figures

Figure S3D

2h 4h 8h 16h BSA Pal

Figure S3F

0 0.1 0.2 0.4 5.5mM Glu 5.5mM 25mM Glu 25mM Diabetes Page 72 of 91

Full unedited blots and microscope images for supplementary figures

Figure S3H

PPARα

PPARγ

PGC1α

pFoxO1

FoxO1

PDK4

β-Tubulin Page 73 of 91 Diabetes

Full unedited blots and microscope images for supplementary figures

Figure S3I

PPARα

PPARγ

PGC1α

pFoxO1

FoxO1

β-Tubulin Diabetes Page 74 of 91

Full unedited blots and microscope images for supplementary figures

Figure S3J

PDK4

PDK2

FoxO1

Β-Tubulin Page 75 of 91 Diabetes

Full unedited blots and microscope images for supplementary figures

Figure S4A

PDK4

IP3R1

GRP75

VDAC1

Mfn2

RyR1

β-Tubulin Diabetes Page 76 of 91

Full unedited blots and microscope images for supplementary figures

Figure S4C & D

Figure S4C Figure S4D

IP3R1-GRP75 IP3R1-VDAC1 BSA siCon BSA siPDK4 BSA siCon BSA siPDK4

Pal siCon Pal siPDK4 Pal siCon Pal siPDK4 Page 77 of 91 Diabetes

Full unedited blots and microscope images for supplementary figures

Figure S4E

PDK4

IP3R1

GRP75

VDAC1

COX IV Diabetes Page 78 of 91

Full unedited blots and microscope images for supplementary figures

Figure S4F & G

Figure S4F

Figure S4G Page 79 of 91 Diabetes

Full unedited blots and microscope images for supplementary figures

Figure S4J

Mfn1

OPA1

p-Drp1s637

p-Drp1s616

Drp1

Fis1

HSP90 Diabetes Page 80 of 91

Full unedited blots and microscope images for supplementary figures

Figure S4K

Mfn1

Mfn2

OPA1

p-Drp1s637

p-Drp1s616

Drp1

Fis1

β-Tubulin Page 81 of 91 Diabetes

Full unedited blots and microscope images for supplementary figures

Figure S4L

WT Pdk4-/- Diabetes Page 82 of 91

Full unedited blots and microscope images for supplementary figures

Figure S5C

Complex V Complex III Complex IV Complex II Complex I

Gapdh Page 83 of 91 Diabetes

Full unedited blots and microscope images for supplementary figures

Figure S5E

IP3R1

GRP78

p-PERK

PERK

CHOP

COX IV Diabetes Page 84 of 91

Full unedited blots and microscope images for supplementary figures

Figure S5F

GRP75

p-IRE1α

IRE1α

p-eIF2α

eIF2α

CHOP

β-Tubulin Page 85 of 91 Diabetes

Full unedited blots and microscope images for supplementary figures

Figure S5G

p-IRE1α

IRE1α

p-JNK1

JNK1

p-eIF2α

eIF2α

CHOP

β-Actin Diabetes Page 86 of 91

Full unedited blots and microscope images for supplementary figures

Figure S5L

siCon siCon siPDK4 Tg Tg

Figure S5M

GRP75

PDK4

CHOP

COX IV Page 87 of 91 Diabetes

Full unedited blots and microscope images for supplementary figures

Figure S6A

IP3R1

p-JNK1

JNK1

β-Tubulin

Figure S6B

p-AKT

AKT

β-Tubulin Diabetes Page 88 of 91

Full unedited blots and microscope images for supplementary figures

Figure S6C

GRP75

p-JNK1

JNK1

COX IV

Figure S6D

p-AKT

AKT

β-Tubulin Page 89 of 91 Diabetes

Full unedited blots and microscope images for supplementary figures

Figure S6E

HA

p-JNK1

JNK1

Figure S6F

p-AKT HA

AKT

β-Tubulin Diabetes Page 90 of 91

Full unedited blots and microscope images for supplementary figures

Figure S6G

PDK4

p-JNK1

JNK1

HSP90

Figure S6H

p-AKT

AKT

β-Actin Page 91 of 91 Diabetes