Antibodies

Rabbit anti-phospho-AMPKα-Thr172 (Cat# 2535, RRID: AB_331250; 1:1000 for immunoblotting (IB)), anti-AMPKα (Cat# 2532, RRID: AB_330331; 1:1000 for IB), anti-phospho-AMPK substrate motif (Cat# 5759, RRID: AB_10949320; 1:1000 for IB and 1:25 for immunoprecipitation (IP)), anti-phospho-ACC-Ser79 (Cat# 3661, RRID: AB_330337; 1:1000 for IB), anti-ACC (Cat# 3662, RRID: AB_2219400; 1:1000 for IB), anti-cytochrome C (Cat# 4280, RRID: AB_10695410; 1:500 for IB), anti-PDI (Cat# 3501, RRID: AB_2156433; 1:1000 for IB), anti-calreticulin (Cat# 12238, RRID: AB_2688013; 1:1000 for IB), anti-erlin2 (Cat# 2959, RRID: AB_2277907; 1:1000 for IB), anti-PDH (Cat# 3205, RRID: AB_2277907; 1:1000 for IB), anti-COXIV (Cat# 4850, RRID: AB_2085424; 1:1000 for IB); anti-14-3-3 (Cat# 95422; 1:1000 for IB), anti-GST-tag (Cat# 2625, RRID: AB_490796; 1:4000 for IB), anti-His-tag (Cat# 12698, RRID: AB_2744546; 1:1000 for IB), anti-Myc-tag (Cat# 2278, RRID: AB_490778; 1:120 for immunofluorescence (IF)), horseradish peroxidase (HRP)-conjugated mouse anti-rabbit IgG (conformation-specific, Cat# 5127, RRID: AB_10892860; 1:2000 for IB), HRP-conjugated goat anti-rat IgG (conformation-specific, Cat# 98164; 1:2000 for IB) and mouse anti-Myc-tag (Cat# 2276, RRID: AB_331783; 1:500 for IB) antibodies were purchased from Cell Signaling Technology. Rabbit anti-calnexin (Cat# ab22595, RRID: AB_2069006; 1:1000 for IB), anti-transferrin (Cat# ab1223, RRID: AB_298951; 1:500 for IB), anti-GLS1 (Cat# ab202027; 1:120 for IF), and mouse anti-CPT1α (Cat# ab128568, RRID: AB_11141632; 1:1000 for IB), mouse anti-total oxidative phosphorylation (OXPHOS) complex (Cat# ab110413, RRID: AB_2629281; 1:1000 for IB) antibodies were purchased from Abcam. Rabbit anti-PDZD8 (Cat# NBP2-58671; 1:1000 for IB or 1:100 for IP; validated in Supplementary information, Fig. S4b) was purchased from Novus Biologicals. Mouse anti-ASCL4 (also known as FACL4; Cat# sc-365230, RRID: AB_10843105; 1:1000 for IB) and anti-HA-tag (Cat# sc-7392, RRID: AB_2894930; 1:1000 for IB, 1:500 for IP or 1:120 for IF) antibodies were purchased from Santa Cruz Biotechnology. Rabbit anti-GLS1 (KGA and GAC; Cat# 12855-1-AP, RRID: AB_2110381; 1:2000 for IB and 1:100 for IP), anti-TOMM20 (Cat# 11802-1-AP, RRID: AB_2207530; 1:1000 for IB), anti-PDK4 (Cat# 12949-1-AP, RRID: AB_2161499; 1:1000 for IB), anti-CPT1β (Cat# 22170-1-AP, RRID: AB_2713959; 1:1000 for IB), anti-PDH E1 alpha (PDHA1; Cat# 18068-1-AP, RRID: AB_2162931; 1:5000 for IB), and mouse anti-tubulin (Cat# 66031-1-Ig, RRID: AB_11042766; 1:20,000 for IB mammalian tubulin) antibodies were purchased from Proteintech. Rabbit anti-APEX2 (Cat# PA5-72607; 1:1000 for IB) antibody was purchased from ThermoFisher Scientific. Mouse anti-FLAG M2 (Cat# F1804, RRID: AB_262044; 1:1000 for IB) antibody was purchased from Sigma. Rabbit anti-RMDN3 (also known as PTPIP51; Cat# A5820, RRID: AB_2766572; 1:1000 for IB) antibody was purchased from Abclonal. The HRP-conjugated goat anti-mouse IgG (Cat# 115-035-003, RRID: AB_10015289; 1:5000 dilution for IB) and goat anti-rabbit IgG (Cat# 111-035-003, RRID: AB_2313567; 1:5000 dilution for IB and 1:120 dilution for IHC) antibodies were purchased from Jackson ImmunoResearch.

Chemicals and assay kits

Aldometanib was synthesized as described previously84 and is now available at MedChemExpress (Cat# HY-148189), GLPBIO (Cat# GC66024), and CymitQuimica (Cat# TM-T60122). Glucose (Cat# G7021), DMSO (Cat# D2650), PBS (Cat# P5493), NaCl (Cat# S7653), KCl (Cat# P9333), HCl (Cat# 320331), ATP (disodium salt; Cat# A6419), ATP (magnesium salt, for kinase assay; Cat# A9187), SDS (Cat# 436143), CaCl2 (Cat# C5670), KH2PO4 (Cat# P5655), K2HPO4 (Cat# P9666), HEPES (Cat# H4034), MES (Cat# 69889), EDTA (Cat# E6758), EGTA (Cat# E3889), MgCl2 (Cat# M8266), CsCl (Cat# 289329), NaAc (Cat# S7670), ethanol (Cat# 459836), glycerol (Cat# G5516), IGEPAL CA-630 (NP-40, Cat# I3021), Triton X-100 (Cat# T9284), Tween-20 (Cat# P9416), cholesteryl hemisuccinate (CHS; Cat# C6512), sodium deoxycholate (Cat# S1827), dithiothreitol (DTT; Cat# 43815), IPTG (Cat# I6758), nuclease-free water (for IVF; Cat# W4502), L-glutathione reduced (GSH; Cat# G4251), mineral oil (Cat# M5310 for IVF, and Cat# M5904 for CsCl density gradient), biotinyl tyramide (biotin-phenol; Cat# SML2135), Trizma base (Tris; Cat# T1503), hexadimethrine bromide (polybrene; Cat# H9268), sodium pyrophosphate (Cat# P8135), β-glycerophosphate (Cat# 50020), hydrogen peroxide (H2O2; Cat# H1009), sodium azide (NaN3; Cat# S2002), sodium ascorbate (Cat# A4034), 6-Hydroxy-2,5,7,8-tetramethylchromane-2-carboxylic acid (Trolox; Cat# 238813), sodium carbonate (Na2CO3; Cat# S7795), O-(carboxymethyl)hydroxylamine hemihydrochloride (AOA; Cat# C13408), urea (Cat# U5378), myristic-d27 acid (Cat# 68698), glutamine (Cat# G8540), carnitine (Cat# C0283), BSA (Cat# A2153), fatty acid-free BSA (Cat# SRE0098), methoxyamine hydrochloride (Cat# 89803), MTBSTFA (with 1% t-BDMCS; Cat# M-108), pyridine (Cat# 270970), sodium palmitate (PA; Cat# P9767), methanol (Cat# 646377), chloroform (Cat# C7559), heparin sodium salt (Cat# H3149), acetonitrile (Cat# 34888), ammonium acetate (Cat# 73594), ammonium hydroxide solution (Cat# 338818), LC-MS-grade water (Cat# 1153332500), mannitol (Cat# M4125), L-methionine sulfone (Cat# M0876), D-campher-10-sulfonic acid (Cat# 1087520), 3-aminopyrrolidine dihydrochloride (Cat# 404624), N,N-diethyl-2-phenylacetamide (Cat# 384011), trimesic acid (Cat# 482749), diammonium hydrogen phosphate (Cat# 1012070500), ammonium trifluoroacetate (Cat# 56865), paraformaldehyde (Cat# 158127), haematoxylin solution (Cat# 03971), eosin Y solution (Cat# 318906), Canada balsam (Cat# C1795), xylene (Cat# 214736), oligomycin A (Cat# 75351), FCCP (Cat# C2920), antimycin A (Cat# A8674), rotenone (Cat# R8875), gentamycin (Cat# 345814), collagenase A (Cat# 11088793001), imidazole (Cat# I5513), taurine (Cat# T8691), ADP (Cat# 01897), phosphocreatine (Cat# V900832), leupeptin (L2884), saponin (Cat# S4521), lactobionate (Cat# L3375), glutamate (Cat# G8415), malate (Cat# M7397), succinate (Cat# S9512), sucrose (Cat# S7903), digitonin (Cat# D141), sodium pyruvate (for Oxygraph-2k measurement; Cat# P5280), formaldehyde solution (formalin; F8775), glutaraldehyde solution (Cat# G5882), glycine (Cat# G8898), K3Fe(CN)6 (Cat# 455946), thiocarbonohydrazide (Cat# 223220), Pb(NO3)2 (Cat# 203580), sodium citrate (Cat# 71497), potassium acetate (Cat# P1190), magnesium acetate (Cat# M5661), MEA (Cat# 30070), glucose oxidase (Cat# G2133), catalase (Cat# C40), OptiPrep (Cat# D1556), Percoll (Cat# P4937), Coomassie Brilliant Blue R-250 (Cat# 1.12553), chymotrypsin (Cat# C3142), formic acid (Cat# 5.43804), β-mercaptoethanol (Cat# M6250), MOPS (Cat# M3183), acetic acid (Cat# 27225), L-glutamic dehydrogenase (GDH; Cat# G2626), NAD+ (Cat# N3014), BPTES (Cat# SML0601), LPS (Cat# L2630), etomoxir (Cat# 236020), human tubal fluid (HTF) medium (Cat# MR-070-D), KSOM medium (Cat# MR-121-D), triple-free DMEM (Cat# D5030), Lysosome Isolation Kit (Cat# LYSISO1), Endoplasmic Reticulum Isolation Kit (Cat# ER0100), Glutamate Assay Kit (Cat# MAK004), anti-FLAG M2 affinity gel (Cat# A2220; 1:500 for IP), FLAG peptide (Cat# F3290), HIS-Select Nickel Affinity Gel (Cat# P6611), and Duolink In Situ Red Starter Kit (Mouse/Rabbit; Cat# DUO92101) were purchased from Sigma. MK-8722 (Cat# HY-111363), R162 (Cat# HY-103096), and compound 968 (Cat# HY-12682) were purchased from MedChemExpress. Torin 1 (Cat# S2827) was purchased from Selleck. Penicillin-streptomycin (Cat# 15140163), Phusion High-Fidelity DNA Polymerase kit (Cat# F530N), mMESSAGE mMACHINE T7 Transcription Kit (Cat# AM1344), MEGAclear Transcription Clean-Up Kit (Cat# AM1908), MEGAshortscript T7 Transcription Kit (Cat# AM1354), SulfoLink Immobilization Kit for Peptides (Cat# 44999), IL-6 Mouse Uncoated ELISA Kit (Cat# 88-7064-88), TNF alpha Mouse Uncoated ELISA Kit (Cat# 88-7324-88), DMEM, high glucose (DMEM; Cat# 11965175), glucose-free DMEM (Cat# 11966025), RPMI 1640 medium (Cat# 11875119), FBS (Cat# 10099141 C), Lipofectamine 2000 (Cat# 11668500), MEM non-essential amino acids solution (Cat# 11140050), GlutaMAX (Cat# 35050061), sodium pyruvate (Cat# 11360070), ProLong Diamond antifade mountant (Cat# P36970), ProLong Live Antifade reagent (Cat# P36975), Streptavidin Magnetic Beads (Cat# 88817; 1:100 for IP), NeutrAvidin agarose (Cat# 29204), EZ-Link Sulfo-NHS-SS-Biotin (Cat# 21331), and Prestained Protein MW Marker (Cat# 26612) were purchased from ThermoFisher Scientific. Red Cell Lysis Buffer (Cat# RT122) was purchased from TIANGEN. OsO4 (Cat# 18465) and uranyl acetate (Cat# 19481) were purchased from Tedpella. Paraplast (Cat# 39602012) was purchased from Leica. SPI-Pon 812 Embedding Kit (Cat# 02660-AB) was purchased from Structure Probe, Inc. n-dodecyl-β-D-maltopyranoside (DDM; Cat# D310) was purchased from Anatrace Products, LLC. Difco LB Broth (Cat# 240220) was purchased from BD. Seahorse XF base medium (Cat# 103334) and Seahorse XF Calibrant solution (Cat# 100840) were purchased from Agilent. Antifade Mounting Medium (Cat# H-1000-10) was purchased from Vector Laboratories, Inc. PrimeSTAR HS polymerase (Cat# R40A) was purchased from Takara. Polyethylenimine (PEI; Cat# 23966) was purchased from Polysciences. Nonfat dry milk (Cat# #9999) and normal goat serum (NGS; Cat# #5425) were purchased from Cell Signaling Technology. Protease inhibitor cocktail (Cat# 70221) was purchased from Roche. WesternBright ECL and peroxide solutions (Cat# 210414-73) were purchased from Advansta. [U-13C]-glutamine (Cat# 184161-19-1), [U-13C]-palmitate ([U-13C]-PA; Cat# CLM-3943), [alpha-15N]-glutamine (Cat# NLM-1016), tryptophan-d5 (Cat# DLM-1092), and [U-13C]-glucose (CLM-1396) were purchased from Cambridge Isotope Laboratories. The isotope-labeled AMP (Cat# 123603801), ADP (Cat# 129603601), and ATP (Cat# 121603801) standards were purchased from Silantes. 3-hydroxynaphthalene-2,7-disulfonic acid disodium salt (2-naphtol-3,6-disulfonic acid disodium salt; Cat# H949580) was purchased from Toronto Research Chemicals. Hexakis(1H,1H,3H-perfluoropropoxy)phosphazene (hexakis(1H,1H, 3H-tetrafluoropropoxy)phosphazine; Cat# sc-263379) was purchased from Santa Cruz Biotechnology. MinElute PCR Purification Kit (Cat# 28004) was purchased from Qiagen. Human chorionic gonadotrophin (hCG) and pregnant mare’s serum gonadotrophin (PMSG) were purchased from Sansheng Biological Technology Co., Ltd. (Ningbo, China). rProtein A Sepharose Fast Flow (Cat# 17127904), Protein G Sepharose 4 Fast Flow (Cat# 17061806), Glutathione Sepharose 4 Fast Flow (Cat# 17513203), and Superdex 200 Increase 10/300 GL (Cat# 28990944) were purchased from Cytiva.

Mouse strains

Protocols for all rodent experiments were approved by the Institutional Animal Care and the Animal Committee of Xiamen University (XMULAC20180028 and XMULAC20220050). WT C57BL/6 J mice (#000664) were obtained from The Jackson Laboratory. AXINF/F and LAMTOR1F/F mice were generated and validated as described previously.38AMPKα1F/F (Cat# 014141), AMPKα2 F/F (Cat# 014142), and RICTORF/F (Cat# 020649) mice were obtained from The Jackson Laboratory, provided by Dr. Sean Morrison. LKB1F/F mice were obtained from Frederick National Laboratory for Cancer Research and provided by Dr. Ronald A. DePinho. PDZD8–/– (KO-first; Pdzd8tm1a(EUCOMM)Wtsi) mice were obtained from Wellcome Trust Sanger Institute, and GLS1F/F mice (#T015195) from GemPharmatech. AMPKα1/2F/F mice were crossed with Mck-Cre mice to generate skeletal muscle-specific knockout (AMPKα-MKO) mice (validated in ref. 84).

To generate mice with muscular PDZD8 replaced with WT PDZD8 or PDZD8-T527A, the PDZD8–/– mice were first crossed with FLPo mice (036512-UCD; MMRRC) to generate the PDZD8F/F mice. WT PDZD8 or its T527A mutant was then introduced to the PDZD8F/F mice under the Rosa26-LSL(LoxP-Stop-LoxP) system,111 followed by crossing with HSA-CreERT2 mice (Cat# 025750; The Jackson Laboratory). The removal of endogenous PDZD8 and the LSL cassette ahead of introduced PDZD8 and PDZD8-T527A (to trigger the expression of introduced PDZD8) was achieved by intraperitoneally injecting mice with tamoxifen (dissolved in corn oil) at 200 mg/kg, 3 times a week. To generate mice with macrophagic PDZD8 being replaced with PDZD8 or PDZD8-T527A, the PDZD8F/F mice carrying PDZD8 or PDZD8-T527A under the Rosa26-LSL(LoxP-Stop-LoxP) cassette were crossed with VAV1-Cre (Cat# 008610; The Jackson Laboratory) mice.

To introduce PDZD8 or PDZD8-T527A into PDZD8F/F mice, cDNA fragments encoding PDZD8 or PDZD8-T527A were inserted into the Rosa26-CTV vector, followed by purification of the plasmids using CsCl density gradient ultracentrifugation method. Some 100 μg of plasmid was then diluted with 500 μL of di-distilled water, followed by concentrating via centrifuge at 14,000× g at room temperature in a 30-kDa-cutoff filter (UFC503096, Millipore) to 50 μL of solution. The solution was diluted with 450 μL of di-distilled water, followed by another two rounds of dilution/concentration cycles. The plasmid was then mixed with 50 μL of di-distilled water to a final volume of 100 μL, followed by mixing with 10 μL of NaAc solution (3 M stock concentration, pH 5.2). The mixture was then mixed with 275 μL of ethanol, followed by incubating at room temperature for 30 min to precipitate plasmid. The precipitated plasmid was collected by centrifuge at 16,000× g for 10 min at room temperature, followed by washing with 800 μL of 75% (v/v) ethanol (in di-distilled water) twice. After evaporating ethanol by placing the plasmid next to an alcohol burner lamp for 10 min, the plasmid was dissolved in 100 μL of nuclease-free water. The plasmid, along with SpCas9 mRNA and the sgRNAs against the mouse Rosa26 locus, were then microinjected into the in vitro fertilized (IVF) embryos of the PDZD8F/F mice. To generate the SpCas9 mRNA, 1 ng of pcDNA3.3-hCas9 plasmid (constructed by inserting the Cas9 fragment released from Addgene Cat# 41815 into the pcDNA3.3 vector; diluted to 1 ng/μL) was amplified using the Phusion High-Fidelity DNA Polymerase kit on a thermocycler (Cat# T100, Bio-Rad) with the following programs: pre-denaturing at 98 °C for 30 s; denaturing at 98 °C for 10 s, annealing at 68 °C for 25 s, then extending at 72 °C for 2 min in each cycle; and final extending at 72 °C for 2 min; cycle number: 33. The following primer pairs were used: 5′-CACCGACTGAGCTCCTTAAG-3′, and 5′-TAGTCAAGCTTCCATGGCTCGA-3′. The PCR product was then purified using the MinElute PCR Purification Kit following the manufacturer’s instructions. The purified SpCas9 PCR product was then subjected to in vitro transcription using the mMESSAGE mMACHINE T7 Transcription Kit following the manufacturer’s instruction (with minor modifications). Briefly, 5.5 μL (300 ng/μL) of SpCas9 PCR product as the template was mixed with 10 μL of 2× NTP/ARCA solution, 2 μL of 10× T7 Reaction Buffer, 0.5 μL of RNase inhibitor, 2 μL of T7 Enzyme Mix, and 4.5 μL of nuclease-free water, followed by incubating at 37 °C for 2 h. The mixture was then mixed with 1 μL of Turbo DNase, followed by incubating at 37 °C for 20 min to digest the template. The mixture was then mixed with 20 μL of 5× E-PAP Buffer, 10 μL of 25 mM MnCl2, 10 μL of 10 mM ATP, 4 μL of E-PAP enzyme, and 36 μL of nuclease-free water, followed by incubating at 37 °C for 20 min for poly(A) tailing. The tailed product was then purified using the MEGAclear Transcription Clean-Up Kit following the manufacturer’s instructions (with minor modifications). Briefly, some 20 μL of tailed RNA was mixed with 20 μL of Elution Solution, followed by mixing with 350 μL of Binding Solution Concentrate. Some 250 μL of ethanol was added to the mixture, then passed the mixture through the Filter Cartridge and washed with 250 μL of Wash Solution twice. The RNA was then eluted with 50 μL of pre-warmed (at 90 °C) Elution Solution. The sgRNAs were prepared as in the SpCas9 mRNA preparation, except that: (a) the gRNA Cloning Vector (Cat# 41824, Addgene) was used as template, and the following programs: pre-denaturing at 98 °C for 30 s; denaturing at 98 °C for 10 s, annealing at 60 °C for 25 s, then extending at 72 °C for 20 s in each cycle; and final extending at 72 °C for 2 min; cycle number: 33; and following primers: 5′-GAAATTAATACGACTCACTATAGGCGCCCATCTTCTAGAAAGACGTTTTAGAGCTAGAAATAGC-3′, and 5′-AAAAGCACCGACTCGGTGCC-3′; were used; (b) in vitro transcription was performed using the MEGAshortscript T7 Transcription Kit, in which the mixture containing: 7.5 μL (100 ng/μL) of purified PCR product, 2 μL of T7 10× T7 Reaction Buffer, 2 μL of T7 ATP solution, 2 μL of T7 CTP solution, 2 μL of T7 GTP solution, 2 μL of T7 UTP solution, 0.5 μL of RNase inhibitor, 2 μL of T7 Enzyme Mix, and 7.5 μL of nuclease-free water was prepared. In addition, the poly(A) tailing assay was not performed.

To perform IVF on the PDZD8F/F mouse strain (according to ref. 112 with modifications), the 4-week-old PDZD8F/F female mice were intraperitoneally injected with PMSG at a dose of 10 U/mouse. At 46 h after the PMSG injection, 10 U/mouse hCG was intraperitoneally injected. At 12 h after the hCG injection, oocytes from the oviducts of female mice, along with sperms from cauda epididymides and vasa deferentia of 16-week-old, proven stud PDZD8F/F male mice, were isolated. To isolate oocytes, oviducts were briefly left on a filter paper, followed by incubating in a human tubal fluid medium (HTF)/GSH drop on an IVF dish (prepared by placing 200 μL of HTF solution supplemented with 125 mM GSH on a 35-mm dish to form a drop, followed by covering the drop with mineral oil and pre-balancing in a humidified incubator containing 5% CO2 at 37 °C for 0.5 h before use). The ampulla was then torn down by forceps, and the cumulus-oocyte masses inside were collected and transferred to another HTF/GSH drop. To isolate sperms, cauda epididymides and vasa deferentia were briefly left on a filter paper, followed by penetrating with a 26 G needle on the cauda epididymides 5 times. Sperms were then released to an HTF drop on sperm capacitation dish (prepared by placing 200 μL of HTF solution on a 35-mm dish to form a drop, followed by covering the drop with mineral oil and pre-balancing in a humidified incubator containing 5% CO2 at 37 °C for 12 h before use) by slightly pressing/squeezing the cauda epididymides, followed by incubating in a humidified incubator containing 5% CO2 at 37 °C for 0.5 h. The capacitated, motile sperms (located on the edge of each HTF drop) were then collected, followed by adding to the oocyte masses soaked in the HTF/GSH drop, 8 μL per drop. The IVF dishes containing oocyte masses and sperms were then cultured in a humidified incubator containing 5% CO2 at 37 °C for 4 h, followed by collecting and washing oocytes in a KSOM drop (freshly prepared by placing 20 μL of KSOM medium on a 35-mm dish to form a drop, followed by covering the drop with mineral oil and pre-balancing in a humidified incubator containing 5% CO2 at 37 °C for 0.5 h) twice. The oocytes were then cultured in an HTF/GSH drop on an IVF dish for another 12 h in a humidified incubator containing 5% CO2 at 37 °C. The presumptive zygotes (in which 2 pronuclei and an extruded, second polar body could be observed) were then picked up. Some 10 pL of DNA mixture comprising Rosa26-CTV-PDZD8 plasmid (20 ng/μL final concentration), SpCas9 mRNA (120 ng/μL final concentration), and Rosa26 sgRNA (100 ng/μL), was microinjected into each of the zygotes, and were cultured in KSOM medium at 37 °C in a humidified incubator containing 5% CO2. At 16 h of culturing, the zygotes/embryos at the two-cell stage were picked up and transplanted into pseudopregnant ICR female mice (8–10 weeks old, > 26 g; prepared by breeding the in-estrus female with a 14-week-old, vasectomized male at a day before the transplantation), 20 zygotes/embryos per mouse, and the offspring carrying the LSL-PDZD8 or LSL-PDZD8-T527A allele was further outcrossed 6 times to C57BL/6 mice before experiments.

The mice with muscular PDZD8 replaced with PDZD8 or its T527A mutant were validated as depicted in Supplementary information, Fig. S6b. For genotyping Rosa26 locus, the following programs: pre-denaturing at 98 °C for 300 s; denaturing at 95 °C for 30 s, annealing at 64 °C for 30 s, then extending at 72 °C for 45 s in each cycle for 5 cycles; denaturing at 95 °C for 30 s, annealing at 61 °C for 30 s, then extending at 72 °C for 45 s in each cycle for 5 cycles; denaturing at 95 °C for 30 s, annealing at 58 °C for 30 s, then extending at 72 °C for 45 s in each cycle for 5 cycles; denaturing at 95 °C for 30 s, annealing at 55 °C for 30 s, then extending at 72 °C for 45 s in each cycle for 5 cycles; and final extending at 72 °C for 10 min; were used. For genotyping other genes and elements, the following programs: pre-denaturing at 95 °C for 300 s; denaturing at 95 °C for 30 s, annealing at 58 °C for 40 s, then extending at 72 °C for 30 s in each cycle; and final extending at 72 °C for 10 min; cycle number: 35; were used. The following primers: 5′-CGCATAACGATACCACGATATCAACAAG-3′ (Primer #1) and 5′-CCGCCTACTGCGACTATAGAGATATC-3′ (Primer #2) for cleaved FRT; 5′-ATCACGACGCGCTGTATC-3′ (Primer #3) and 5′-ACATCGGGCAAATAATATCG-3′ (Primer #4) for LacZ; 5′-ACTGTCTGTCCTTCCAGGGG-3′ (Primer #5) and 5′-GTGGAAAAGCCAAGAAAGGC-3′ (Primer #6) for LoxP; 5′-GCCACCTTCATGAGCTACAACACC-3′ and 5′-AACAGGAACTGGTACAGGGTCTTGG-3′ for FLPo; 5′-CAGGTAGGGCAGGAGTTGG-3′ and 5′-TTTGCCCCCTCCATATAACA-3′ for HSA-Cre; 5′-AGTGGCCTCTTCCAGAAATG-3′ and 5′-TGCGACTGTGTCTGATTTCC-3′ for the control of HSA-Cre; 5′-TCTCCCAAAGTCGCTCTGAG-3′, 5′-AAGACCGCGAAGAGTTTGTC-3′, and 5′-ATGCTCTGTCTAGGGGTTGG-3′ for Rosa26, 5′-GGAGTTCTATTAAGACGGTTG-3′ and 5′-GTGCTGGGTCTGTTATCTC-3′ for generating PCR products for sequencing T527. The mice with macrophagic PDZD8 replaced with PDZD8 or PDZD8-T527A were validated as described above (depicted in Supplementary information, Fig. S7a), except that VAV1-Cre (5′-GGTGTTGTAGTTGTCCCCACT-3′ and 5′-CAGGTTTTGGTGCACAGTCA-3′ for VAV1-Cre-#1; 5′-AGATGCCAGGACATCAGGAACCT-3′ and 5′-ATCAGCCACACCAGACACAGAGATC-3′ for VAV1-Cre-#2), rather than HSA-Cre, were genotyped.

The following ages of mice were used: (1) for analyzing AMPK activation: WT and AMPKα-MKO mice, 4 weeks old; (2) for analyzing glutaminolysis and FAO in the liver and skeletal muscle tissues: WT mice, AMPKα-MKO mice, and mice with muscular PDZD8 replaced with WT PDZD8 or PDZD8-T527A, aged 10 weeks; (3) for analyzing OCR in mouse skeletal muscles: WT mice, AMPKα-MKO mice, and mice with muscular PDZD8 replaced with WT PDZD8 or PDZD8-T527A, aged 8 weeks; (4) for determining the LPS-induced inflammatory responses, mice with macrophage PDZD8 replaced with WT PDZD8 or PDZD8-T527A, aged 8 weeks.

Cell lines and viruses

In this study, no cell line used is on the list of known misidentified cell lines maintained by the International Cell Line Authentication Committee (https://iclac.org/databases/cross-contaminations/). HEK293T cells (Cat# CRL-3216) were purchased from ATCC. HEK293T cells and MEFs were maintained in DMEM supplemented with 10% FBS, 100 IU penicillin, 100 mg/mL streptomycin at 37 °C in a humidified incubator containing 5% CO2. All cell lines were verified to be free of mycoplasma contamination. HEK293T cells were authenticated by STR sequencing. PEI at a final concentration of 10 μM was used to transfect HEK293T cells. The total DNA to be transfected for each plate was adjusted to the same amount by using the relevant empty vector. Transfected cells were harvested at 24 h after transfection.

Lentiviruses, including those for knockdown or stable expression, were packaged in HEK293T cells by transfection using Lipofectamine 2000. At 30 h post-transfection, medium (DMEM supplemented with 10% FBS and MEM non-essential amino acids; approximately 2 mL) was collected and centrifuged at 5000× g for 3 min at room temperature. The supernatant was mixed with 10 μg/mL polybrene, and was added to MEFs or HEK293T cells, followed by centrifuging at 3000× g for 30 min at room temperature (spinfection). Cells were incubated for another 24 h (MEFs) or 12 h (HEK293T cells) before further treatments.

AMPKα–/– MEFs, RAPTOR–/– MEFs, and AMPKα–/– HEK293T cells were generated and validated as described previously.113LAMTOR1F/F, AXINF/F, and LKB1F/F MEFs were established by introducing SV40 T antigen via lentivirus into cultured primary embryonic cells from mouse litters as described previously,38 so does GLS1F/F and RICTORF/F MEFs. LAMTOR1–/–, AXIN–/–, LKB1–/–, GLS1–/– and RICTOR–/– MEFs were generated by infecting each of MEFs with adenoviruses expressing the Cre recombinase (Cat# 1045, Vector Biolabs) for 12 h. The infected cells were then incubated in the fresh DMEM for another 12 h before further treatments. The GLS1 gene (encoding both KGA and GAC) was knocked down and validated in MEFs as described previously.114 The sequence of siRNA used to knockdown mouse RMDN3 is 5′-GAAGCCGACAAGACTTTCT-3′.

The mouse genes (PDZD8, RMDN3, PDHA1, CPT1A, and CPT1B) were deleted from MEFs using the CRISPR-Cas9 system. Nucleotides were annealed to their complements containing the cloning tag AAAC, and inserted into the back-to-back BsmBI restriction sites of the lentiCRISPRv2 vector (#52961, Addgene). The sequence for each sgRNA is as follows: 5′-CACCCCTCGGCGCCGCCGCCATAA-3′ for PDZD8; 5′-TCTTATGGCGCTGCGGCGCG-3′ for RMDN3; 5′-GCTGTATCCCGCGTGTTGGC-3′ for PDHA1; 5′-GGCGGAGATCGATGCCATCA-3′ for CPT1A; and 5′-TCCACCGGAGTCTGGGCGAC-3′ for CPT1B. The constructs were then subjected to lentivirus packaging using HEK293T cells that were transfected with 2 µg of DNA in Lipofectamine 2000 transfection reagent per well of a 6-well plate. At 30 h post-transfection, the virus (approximately 2 mL) was collected to infect MEFs as described above, except cells cultured to 15% confluence were incubated with the virus for 72 h. When cells approached confluence, they were single-cell sorted into 96-well dishes. Clones were expanded and evaluated for knockout status by sequencing.

For glucose starvation, cells were rinsed twice with PBS and then incubated in glucose-free DMEM supplemented with 10% FBS and 1 mM sodium pyruvate for desired periods at 37 °C.

Data reporting

The chosen sample sizes were similar to those used in this field: n = 3–10 samples to evaluate the levels of metabolites in cells and tissues6,8,37,62,84; n = 3–9 samples to determine OCR in cells and tissues,84,115n = 3–4 samples to determine the activity of GLS1 (ref. 73,116,117); n = 3–4 samples to determine the expression levels and phosphorylation levels of a specific protein in animal cells or tissues38; n = 20–27 cells to determine protein interaction by FRET-FLIM assay in living cells118; n = 14–17 cells to determine protein interaction by PLA119,120; n = 15 to determine the septic deaths and n = 3–4 to determine cytokines.121 No statistical methods were used to predetermine the sample size. All experimental findings were repeated as stated in figure legends, and all additional replication attempts were successful. For animal experiments, mice were maintained under the same condition or place. For cell experiments, cells of each genotype were cultured in the same CO2 incubator and were parallel-seeded and randomly assigned to different treatments. Each experiment was designed and performed along with controls, and samples for comparison were collected and analyzed under the same conditions. Randomization was applied wherever possible. For example, during mass spectrometry analyses (metabolites and proteins), samples were processed and applied to the mass spectrometer in random orders. For animal experiments, sex-matched and age-matched litter-mate animals in each genotype were randomly assigned to different treatments. Otherwise, randomization was not performed. For example, when performing immunoblotting, samples needed to be loaded in a specific order to generate the final figures. Blinding was applied wherever possible. For example, samples or cages during sample collection and processing were labeled as code names that were later revealed by the individual who picked and treated animals or cells, but did not participate in sample collection and processing, until assessing the outcome. Similarly, during microscopy data collection and statistical analyses, the fields of view were chosen randomly and performed by different operators, preventing potentially biased selection for desired phenotypes. Otherwise, blinding was not performed, such as the measurement of GLS1 activity in vitro, as different reagents were added for particular reactions.

Starvation and LPS treatments of mice

Unless stated otherwise, mice were housed with free access to water and a standard diet (65% carbohydrate, 11% fat, 24% protein) under specific pathogen-free conditions. The light was on from 8:00 to 20:00, with the temperature kept at 21–24 °C and humidity at 40%–70%. Only male mice were used in the study, and male littermate controls were used throughout the study.

For starvation, mice were individually caged for 1 week before each treatment. The diet was withdrawn from the cage at 17:00, and mice were sacrificed at desired time points by cervical dislocation. For LPS treatment, mice were intraperitoneally injected with LPS at 10 mg/kg as described previously.122 BPTES (oral gavage, 12.5 mg/kg; freshly prepared by dissolving BPTES powder in DMSO to form a 37.5 mg/mL solution, and then diluted to 1.25 mg/mL in saline), compound 968 (intraperitoneal injection, 10 mg/kg; freshly prepared by dissolving compound 968 powder in DMSO to form a 10 mg/mL solution, and then diluted to 1 mg/mL in saline) or aldometanib (oral gavage, 2 mg/kg; prepared as described previously84) was administered 30 min before LPS injection. Survival curves were covered up to three weeks after injection to ensure that the deaths at the late stage were not omitted. Levels of pro-inflammatory cytokines and lung damage were assessed in separate batches/sets of mice from those used for assessing survival.

Serology

Blood glucose was measured through tail vein bleeding using the OneTouch UltraVue automatic Glucometer (LifeScan). For measuring insulin levels, approximately 100 μL of blood was collected (from the submandibular vein plexus) and was placed at room temperature for 20 min, followed by centrifugation at 3000× g for 10 min at 4 °C. Some 50 μL of the resultant serum was used to determine the levels of insulin using the Mouse or Rat Ultrasensitive Insulin ELISA kit according to the manufacturer’s instructions. The five-parameter logistic fitted standard curve for calculating insulin concentrations was generated from the website of Arigo Biolaboratories (https://www.arigobio.cn/ELISA-calculator/). For measuring free fatty acids, glycerol, β-hydroxybutyrate and glucagon, some 1.3, 10, 1 and 5 μL of serum were used, using the LabAssay NEFA kit, Free Glycerol Assay kit, Ketone Body Assay kit, and Mouse Glucagon ELISA Kit, respectively, all following the manufacturer’s instructions. For measuring TNFα and IL-6, some 1 μL (for IL-6) or 100 μL (for TNFα) of serum, or 100 μL of BMDM culture supernatant, was diluted to 1 mL with PBS, followed by taking 100 μL of solution to measure the cytokine levels following the manufacturer’s instructions.

Histology

To measure the TAG content, mice were euthanized, and the gastrocnemius muscle was immediately removed and rinsed in PBS 3 times. Approximately 100 mg of tissue was homogenized in 1 mL of PBS containing 5% (v/v) Triton X-100. The homogenates were boiled for 5 min, followed by centrifugation at 20,000× g at 25 °C for 10 min. The TAG content (from the 3 μL of supernatant) was determined using Labassay triglyceride reagent.

Muscular glycogen contents were determined using the Glycogen Assay Kit according to the manufacturer’s instructions. Briefly, the mouse gastrocnemius muscle was quickly excised and immediately frozen in liquid nitrogen. Some 100 mg of muscle tissue was homogenized in 1 mL of double-distilled water on ice, then boiled for 5 min, followed by centrifugation at 20,000× g for 10 min. Then, 10 μL of supernatant was mixed with 40 μL of hydrolysis buffer, and mixed with 2 μL of hydrolysis enzyme mix, followed by incubation for 30 min at room temperature. Next, 50 μL of the master reaction mix (through mixing 46 μL of development buffer, 2 μL of development enzyme mixed with 2 μL of fluorescent peroxidase substrate) was added to the sample, followed by incubation for 30 min at room temperature in the dark. The OD570 was then recorded by a SpectraMax M5 microplate reader (Molecular Devices) using the SoftMax Pro software (v.5.4.1.1, Molecular Devices; and same hereafter), and the contents of glycogen were then calculated according to the standard curves generated with Glycogen Standards.

For H&E staining of lung tissues, tissues excised from blood-drained mice were cut into pieces, and were fixed in 4% (v/v) paraformaldehyde for 24 h at room temperature, then transferred to embedding cassettes. The cassettes were then washed in running water for 12 h, followed by successive soaking each for 1 h in 50% ethanol (v/v in water), 30 min in 70% ethanol, and 20 min in 95% ethanol twice. The fixed tissues were further dehydrated in anhydrous ethanol for 15 min twice, followed by immersing in 50% xylene (v/v in ethanol) for 5 min, two changes of xylene, 30 min each; and two changes of paraffin wax (Paraplast; 56 °C), 1.5 h each. The dehydrated tissues were embedded in paraffin on a HistoCore Arcadia Paraffin Embedding Machine (Leica). Paraffin blocks were then sectioned at a thickness of 5 μm, dried on an adhesion microscope slide, followed by rehydrating in the following order: three changes of xylene at 70 °C, 5 min each; two changes of anhydrous ethanol, 2 min each; two changes of 95% ethanol, 1 min each; two changes each for 1 min of 95% ethanol, and then briefly in water. The sections were then stained in hematoxylin solution for 1 min, washed in running water for 10 min, differentiated in 1% HCl (in ethanol) for 30 s, washed in running water for 1 min, immersed in 0.2% (v/v in water) ammonium hydroxide solution for 30 s, washed in running water for 1 min, and stained in eosin Y-solution for 1 min. The stained sections were dehydrated twice in 95% ethanol, 1 min each; twice in anhydrous ethanol, 1 min each; and two changes of xylene, 1 min each. The stained sections were mounted with Canada balsam and visualized on a Leica DM4 B microscope.

Isolation and differentiation of BMDMs

BMDMs were isolated and differentiated as described previously,123 with modifications. Briefly, mice at 4 weeks old were anesthetized, followed by isolating the femur and tibia from the lower limbs by removing the surrounding muscles. After removing both the proximal and distal epiphyses of the femur and tibia, some 3 mL of PBS was injected into the medullary cavity via a 23 G needle from one end of each shaft, and the bone marrow of the other end was collected in a 50-mL conical tube on ice. The bone marrow suspension was then filtered by passing through a 70-μm strainer (BD Falcon), followed by centrifuge at 200× g, 5 min at 4 °C. The cell pellet was then resuspended in 5 mL of Red Cell Lysis Buffer for 5 min, followed by mixing with another 5 mL of RPMI 1640 medium, and then centrifuged at 500 × g, 5 min at 4 °C. The pellet was then resuspended in 10 mL of RPMI 1640 medium supplied with 10% inactivated FBS (heated at 56 °C, 30 min), 30% L929 conditioned medium (prepared as described previously124), penicillin, and streptomycin, and was cultured at 37 °C, 5% CO2 for 7 days, with culture medium being refreshed on day 3, for differentiation. The medium of differentiated BMDMs was then changed to RPMI 1640, supplied with 10% inactivated FBS before further uses.

For in vitro LPS stimulation, BMDM cultured in 6-well plates with a density of 2 × 106 cells per well, were treated with 10 ng/mL LPS under desired glucose concentrations. After 6 h of LPS treatment, the culture supernatant was collected to detect cytokines, and the cells were lysed for immunoblotting.

Generation of the antibody against p-T527-PDZD8

Rabbit polyclonal antibody against p-T527-PDZD8 (1:1,1000 dilution for IB) was raised using the peptide CPLSHSPKRTP(p-T)TLSI (amino acid residues 511–525 of human PDZD8) conjugated to the KLH immunogen (linked to the cysteine residue). A rabbit was then biweekly immunized with 100 µg of KLH-conjugated antigen, which is pre-incubated with 500 µg manganese adjuvant (kindly provided by Dr. Zhengfan Jiang from Peking University, see ref. 125) for 5 min and then mixed with PBS to a total volume of 500 µL, for 4 times, followed by collecting antiserum. The p-T527-PDZD8 antibody was then purified from the antiserum using the CPLSHSPKRTP(p-T)TLSI peptide-conjugated SulfoLink Coupling resin/column supplied in the SulfoLink Immobilization Kit. To prepare the column, 1 mg of the peptide was first dissolved with 2 mL of Coupling Buffer, followed by adding 0.1 mL of TCEP (25 mM stock concentration), then incubated at room temperature for 30 min. The mixture was then incubated with SulfoLink Resin in a column, which was pre-calibrated by 2 mL of Coupling Buffer twice on a rotator at room temperature for 15 min, followed by incubation at room temperature for 30 min without rotating. The excess peptide was then removed, and the resin was washed with 2 mL of Wash Solution 3 times, followed by 2 mL of Coupling Buffer 2 times. The nonspecific-binding sites on the resin were then blocked by incubating with 2 mL of cysteine solution (by dissolving 15.8 mg of L-cysteine-HCl in 2 mL of Coupling Buffer to make a concentration of 50 mM cysteine) on a rotator for 15 min at room temperature, followed by incubating for another 30 min without rotating at room temperature. After removing the cysteine solution, the resin was washed with 6 mL of Binding/Wash Buffer, followed by incubating with 2 mL of antiserum mixed with 0.2 mL of Binding/Wash Buffer for 2 h on a rotator. The resin was then washed with 1 mL of Binding/Wash Buffer for 5 times, and the antibody was eluted with 2 mL of Elution Buffer. The eluent was then mixed with 100 μL of Neutralization Buffer. The antibody against basal PDZD8 in the crude antibody eluent was then removed through a previously described membrane-based affinity purification method.126 Briefly, the bacterially purified, GST-tagged PDZD8-511–525 was subjected to SDS-PAGE, followed by transferring to a PVDF membrane. The PDZD8-bound-membrane was incubated in 5% (w/v) non-fat milk dissolved in TBST (40 mM Tris, 275 μM NaCl, 0.2% (v/v) Tween-20, pH 7.6) for 2 h, then incubated with the crude antibody preparation for 2 days, and then repeated for another 2 times. Antibody was validated for immunoblotting as shown in Supplementary information, Fig. S4g.

Plasmids

All materials generated in this study, including mouse strains, cell lines, and antibodies, are available upon request. The expression plasmids constructed in this study have been deposited to Addgene (https://www.addgene.org/Sheng-cai_Lin/).

Full-length cDNAs used in this study were obtained either by PCR using cDNA from MEFs, or by purchasing from Origene or Sino Biological. Mutations of PDZD8 and GLS1 were performed by PCR-based site-directed mutagenesis using PrimeSTAR HS polymerase. Expression plasmids for various epitope-tagged proteins were constructed in the pcDNA3.3 vector for transfection (ectopic expression in mammalian cells), in the pBOBI vector for lentivirus packaging (stable expression in mammalian cells), in the pLVX-IRES for doxycycline-inducible expression, or the pET-28a and pGEX4T-1 (bacterial expression) vectors. PCR products were verified by sequencing (Invitrogen, China). The lentivirus-based vector pLV-H1-EF1a-puro (for GLS1) was used for the expression of siRNA in MEFs. All plasmids used in this study were purified by the CsCl density gradient ultracentrifugation method.

IP and IB assays

To determine the interaction between endogenous GLS1 and PDZD8, four 10-cm dishes of MEFs (grown to 80% confluence) were collected for each experiment. Cells, starved or unstarved, were lysed with 500 μL per dish of ice-cold DDM/CHS lysis buffer (20 mM HEPES, pH 7.4, 50 mM NaCl, 10 mM MgCl2, 0.5% (w/v) DDM, 0.1% (w/v) CHS with protease inhibitor cocktail) without pre-washing with PBS (same hereafter for all IP and IB assays), followed by sonication and centrifugation at 4 °C for 15 min. Cell lysates were incubated with GLS1 or PDZD8 antibody overnight. Overnight protein aggregates were pre-cleared by centrifugation at 20,000× g for 10 min, and protein A/G beads (1:200 dilution, balanced with DDM/CHS lysis buffer) were then added into the lysate/antibody mixtures for another 3 h at 4 °C. The beads were centrifuged and washed with 100 times the volume of ice-cold DDM/CHS wash buffer (20 mM HEPES, pH 7.4, 50 mM NaCl, 10 mM MgCl2, 0.01% (w/v) DDM, 0.002% (w/v) CHS) 3 times (by centrifuging at 2000× g) at 4 °C and then mixed with an equal volume of 2× SDS sample buffer and boiled for 10 min before IB.

To determine the interaction between ectopically expressed GLS1 and PDZD8, a 6-cm dish of HEK293T cells were transfected with different expression plasmids. At 24 h after transfection, cells were collected and lysed in 500 µL of ice-cold DDM/CHS lysis buffer, followed by sonication and centrifugation at 4 °C for 15 min. Anti-HA-tag (1:100) or anti-Myc-tag (1:100) antibodies, along with protein A/G beads (1:100), or anti-FLAG M2 Affinity Gel (1:200, pre-balanced in DDM/CHS lysis buffer) was added into the supernatant and mixed for 4 h at 4 °C. The beads were washed with 200 times the volume of DDM/CHS wash buffer 3 times at 4 °C, mixed with an equal volume of 2× SDS sample buffer, and boiled for 10 min before immunoblotting.

To determine the interaction between stably expressed FLAG-tagged PDZD8 and endogenous 14-3-3, one 10-cm dish of MEFs (grown to 80% confluence) were collected for each experiment. IP was performed as in determining the interaction between ectopically expressed GLS1 and PDZD8, except that ice-cold Triton lysis buffer (20 mM Tris-HCl, pH 7.5, 150 mM NaCl, 1 mM EDTA, 1 mM EGTA, 1% (v/v) Triton X-100, 2.5 mM sodium pyrophosphate, 1 mM β-glycerophosphate, with protease inhibitor cocktail) was used to lyse cells and wash ANTI-FLAG M2 Affinity Gel. The M2 Affinity Gel was eluted with 30 μL of FLAG Peptide (400 μg/mL final concentration) for another 45 min at 4 °C. Some 30 μL of eluent was then collected, mixed with 7.5 μL of 5× SDS buffer, and boiled for 10 min before immunoblotting.

To verify the phosphorylation of MAM proteins (listed in Supplementary information, Table S1) by AMPK (using the anti-pan-phospho-AMPK substrate antibody), a 10-cm dish of HEK293T cells were transfected with different expression plasmids. IP was performed as in determining the interaction between ectopically expressed GLS1 and PDZD8, except that ice-cold Triton lysis buffer was used to lyse cells and wash protein A/G beads. In particular, antibodies were incubated with cell lysates for a time duration of 15 min to avoid the possible phosphorylation mediated by AMPK in the lysate (even in the unstarved cells).

The APEX2 proximity labeling assay was performed as described previously,127 with minor modifications. Briefly, protein biotinylation reactions in 60 10-cm dishes of MEFs with stable expression of APEX2-PDZD8 were treated with DMEM (10 mL per dish) containing 500 mM biotinyl tyramide at 37 °C for 30 min, followed by the addition of 1 mM hydrogen peroxide and incubated at room temperature for 1 min. The reactions were then terminated by removing the medium and adding ice-cold quenching buffer (10 mM sodium azide, 10 mM sodium ascorbate, 5 mM Trolox, in PBS), 10 mL per dish. Cells were washed with PBS, 10 mL per dish, followed by subcellular fractionation (see “Subcellular fractionation” section). Each fraction was lysed with 500 µL of ice-cold RIPA buffer (50 mM Tris-HCl, pH 7.5, 150 mM NaCl, 1% NP-40, 0.5% sodium deoxycholate, 0.1% SDS, with protease inhibitor cocktail), and was centrifuged at 20,000× g for 10 min at 4 °C. The supernatant was mixed with 20 μL of Streptavidin Magnetic Beads for 12 h on a rotator at 4 °C, then washed with 1 mL of ice-cold RIPA buffer twice. The beads were then washed with ice-cold RIPA buffer supplemented with 1 M KCl once, ice-cold RIPA buffer supplemented with 0.1 M Na2CO3 once, 2 M ice-cold urea dissolved in 10 mM Tris-HCl, pH 8.0 once, and ice-cold RIPA buffer twice. The beads slurry was then mixed with an equal volume of 2× SDS sample buffer and boiled for 10 min before immunoblotting.

To analyze the levels of p-AMPKα, p-ACC, and p-PDZD8 in MEFs or macrophages, cells grown to 70%–80% confluence in a well of a 6-well dish were lysed with 250 μL of ice-cold Triton lysis buffer. The lysates were then centrifuged at 20,000× g for 10 min at 4 °C, and an equal volume of 2× SDS sample buffer was added into the supernatant. Samples were then boiled for 10 min and then directly subjected to immunoblotting. To analyze the levels of p-AMPKα, p-ACC, and p-PDZD8 in muscle, liver, and heart tissues, mice were anesthetized after indicated treatments. Freshly excised (or freeze-clamped) tissues were immediately lysed with ice-cold Triton lysis buffer (10 μL/mg tissue weight for liver and 5 μL/mg tissue weight for skeletal muscle and heart), followed by homogenization and centrifugation as described above. The lysates were then mixed with 2× SDS sample buffer, boiled, and subjected to immunoblotting. All samples were subjected to immunoblotting on the same day of preparation, and any freeze-thaw cycles were avoided.

For immunoblotting, the SDS-polyacrylamide gels were prepared in-house. The thickness of the gels used in this study was 1.0 mm. Samples of less than 10 μL were loaded into wells, and the electrophoresis was run at 100 V (by PowerPac HC High-Current Power Supply, Bio-Rad) in a Mini-PROTEAN Tetra Electrophoresis Cell (Bio-Rad). In this study, all samples were resolved on 8% resolving gels, except that PDZD8-CT was run on 10% gels, and those smaller than 35 kDa including TOMM20, cytochrome C, ERLIN2 and some PDZD8 truncations on 15% gels. The resolved proteins were then transferred to the PVDF membrane (0.45 μm, Cat# IPVH00010, Merck). The PVDF membrane was then blocked by 5% (w/v) BSA (for all antibodies against phosphorylated proteins) or 5% (w/v) non-fat milk (for all antibodies against total proteins) dissolved in TBST for 2 h on an orbital shaker at 60 rpm at room temperature, followed by rinsing with the TBST for twice, 5 min each. The PVDF membrane was then incubated with a primary antibody overnight at 4 °C on an orbital shaker at 60 rpm, followed by rinsing with TBST 3 times, 5 min each at room temperature, and then the secondary antibodies for 3 h at room temperature with gentle shaking. The secondary antibody was then removed, and the PVDF membrane was further washed with TBST 3 times, 5 min each, at room temperature. PVDF membrane was incubated in an ECL mixture (by mixing equal volumes of ECL solution and Peroxide solution for 5 min), then life with Medical X-Ray Film (FUJIFILM). The films were then developed with X-OMAT MX Developer (Carestream) and X-OMAT MX Fixer and Replenisher solutions (Carestream) on a Medical X-Ray Processor (Carestream) using Developer (Model 002, Carestream). The developed films were scanned using a Perfection V850 Pro scanner (Epson) with an Epson Scan software (v.3.9.3.4), and were cropped using Photoshop 2023 software (Adobe). Levels of total proteins and phosphorylated proteins were analyzed on separate gels, and representative immunoblots were shown. Uncropped immunoblots are uploaded as a “Full scans