mTORC2–NDRG1–CDC42 axis couples fasting to mitochondrial fission

Fasting triggers diverse physiological adaptations including increases in circulating fatty acids and mitochondrial respiration to facilitate organismal survival. The mechanisms driving mitochondrial adaptations and respiratory sufficiency during fasting remain incompletely understood. Here we show that fasting or lipid availability stimulates mTORC2 activity. Activation of mTORC2 and phosphorylation of its downstream target NDRG1 at serine 336 sustains mitochondrial fission and respiratory sufficiency. Time-lapse imaging shows that NDRG1, but not the phosphorylation-deficient NDRG1Ser336Ala mutant, engages with mitochondria to facilitate fission in control cells, as well as in those lacking DRP1. Using proteomics, a small interfering RNA screen, and epistasis experiments, we show that mTORC2-phosphorylated NDRG1 cooperates with small GTPase CDC42 and effectors and regulators of CDC42 to orchestrate fission. Accordingly, RictorKO, NDRG1Ser336Ala mutants and Cdc42-deficient cells each display mitochondrial phenotypes reminiscent of fission failure. During nutrient surplus, mTOR complexes perform anabolic functions; however, paradoxical reactivation of mTORC2 during fasting unexpectedly drives mitochondrial fission and respiration.

Fasting triggers diverse physiological adaptations including increases in circulating fatty acids and mitochondrial respiration to facilitate organismal survival. The mechanisms driving mitochondrial adaptations and respiratory sufficiency during fasting remain incompletely understood. Here we show that fasting or lipid availability stimulates mTORC2 activity. Activation of mTORC2 and phosphorylation of its downstream target NDRG1 at serine 336 sustains mitochondrial fission and respiratory sufficiency. Time-lapse imaging shows that NDRG1, but not t he p ho sp ho ry la ti on -d eficient NDRG1 Ser336Ala mutant, engages with mitochondria to facilitate fission in control cells, as well as in those lacking DRP1. Using proteomics, a small interfering RNA screen, and epistasis experiments, we show that mTORC2-phosphorylated NDRG1 cooperates with small GTPase CDC42 and effectors and regulators of CDC42 to orchestrate fission. Accordingly, Rictor KO , NDRG1 Ser336Ala mutants and Cdc42-deficient cells each display mitochondrial phenotypes reminiscent of fission failure. During nutrient surplus, mTOR complexes perform anabolic functions; however, paradoxical reactivation of mTORC2 during fasting unexpectedly drives mitochondrial fission and respiration.
Dynamic mitochondrial networks are essential for mitochondrial function and organismal wellbeing 1 . Mitochondrial networking is in turn controlled by coordinated fission and fusion events regulated by proteins localized at endoplasmic reticulum (ER)-mitochondria contacts (mitochondria-associated membranes, MAMs) 2 . Indeed, blocking fission by deleting dynamin-related protein 1 (DRP1) (refs. 3,4) or mitochondrial fission factor (MFF) 4 or inhibiting fusion by silencing optic atrophy 1 (OPA1) (ref. 5) and mitofusin (MFN) proteins 6 alters mitochondrial morphology and function. Although, recent work has identified a role of AMPK in mitochondrial fission 7 , we do not completely understand how dietary stressors such as fasting influence mitochondrial dynamics in intact organisms. Since nutrient signalling is coupled to healthspan, it remains critical to understand how impairment in these processes lead to age-related diseases.
In this Article, we show that nutrient-responsive mTORC2 is paradoxically reactivated by fasting to stimulate mitochondrial fission.

mTORC2 supports mitochondrial respiration during fasting
To determine the physiological roles of mTOR reactivation during fasting, we inactivated mTORC1 or mTORC2 or hyperactivated mTORC1 by knocking out Raptor 21 , Rictor 22 or Tsc1 (ref. 23), respectively, using liver-restricted AAV8-TBG-iCre (Fig. 2a,b and Extended Data Fig. 3a,b). Loss of Rictor/mTORC2 activity in liver, and not fat or muscle, was confirmed by reduced AKT Ser473 phosphorylation (Extended Data Fig. 3c). Since fasted livers accumulate triglyceride, we examined the effect of We show that the mTORC2-SGK1 cascade phosphorylates a known target NDRG1 (ref. 8) at Ser336, which then engages with mitochondria to drive fission. NDRG1, but not the phosphorylation-deficient NDRG1 Ser336Ala mutant, interacts with CDC42 (ref. 9), a cytokinetic protein with intrinsic GTP hydrolysis activity 10 , to drive fission. mTORC2, NDRG1 and CDC42 each localize to MAMs, and silencing Rictor, Ndrg1 or Cdc42 or identified CDC42 effectors blocks fission. Thus, paradoxical reactivation of an mTORC2-NDRG1 Ser336 -CDC42 axis drives mitochondrial fission during fasting.

Fasting or lipid availability activates mTORC1/2 signalling
Fasting increases circulating free fatty acids (FFAs), which undergo mitochondrial oxidation to support organismal sustenance 11 . To understand the mechanisms driving metabolic adaptations during fasting or fatty acid availability, we sought to identify the signalling cascades that are activated under these conditions. To this purpose, we performed unbiased quantitative phosphoproteomics in livers of mice that were (1) basal fed; (2) overnight (14-16 h) fasted; or fasted overnight and then gavaged with (3) dietary triglycerides as corn oil; or (4) BODIPY FL C 16 /palmitic acid; or (5) refed a high-fat diet (Fig. 1a). Corn oil or BODIPY FL C 16 groups served as models for exogenous lipid availability, while refeeding served as a control to simulate physiological feeding. Corn oil is absorbed as FFA and repackaged and secreted by enterocytes as lipoproteins and subsequently delivered to liver as FFA. Delivery of BODIPY FL C 16 to livers was confirmed by direct fluorescence of liver slices (Extended Data Fig. 1a). Phosphoproteomics in the five groups identified 2,160 phosphosites across 942 phosphoproteins, of which 863 phosphosites (39.95%) were significantly modulated. Unsupervised hierarchical clustering analyses grouped basal and refed cohorts into one cluster, while lipid-exposed groups (that is, fasted, corn oil and BODIPY FL C 16 ) clustered into the second group (Extended Data Fig. 1b and Supplementary Table 1). A second clustering analysis to determine phosphoproteins that are coordinately modulated revealed a major 'green cluster' encompassing 86.9% of significantly modulated phosphosites (Fig. 1b and Supplementary Table 2). The average normalized abundance of phosphosites belonging to the green cluster (to better appreciate group-to-group modulation rather than phosphoprotein expression differences) was significantly higher in lipid-exposed groups, when compared with basal and refed groups (Fig. 1c). Interestingly, despite strong reduction in phosphorylations in the refed group, the green cluster-normalized abundance was relatively higher in the refed cohort compared with the basal fed cohort, indicating qualitative differences in phosphopeptides between basal fed and refed groups (Fig. 1c).
To predict the kinases putatively modulating the phosphosites in the green cluster, we used GPS algorithm with the interaction filter, or in vivo GPS (iGPS) 12 , which revealed that these phosphosites are targets of cyclin-dependent kinases (CDKs), Ca 2+ /calmodulin-dependent kinases, mitogen-activated protein kinases (MAPKs) and Ser/threonine a, Phosphoproteomics in livers as per plan in cartoon. b, Heat map and hierar chical clustering of phosphosites across groups indicated in a. c, Phosphoproteomewide comparisons via z score normalization of phosphosites in green cluster. Grey dots represent individual phosphosites. Blue diamonds represent group means. ***P < 0.001, non-parametric ANOVA (Kruskal-Wallis statistic 797.3, P < 0.0001) followed by Dunn's multiple comparisons test. d, iGPS prediction of upstream individual kinases that respond to lipids across groups indicated and identified in the green cluster in b. e-g, Pairwise comparisons between indicated groups (fasted versus basal (e), corn oil versus fasted (f), and BODIPY FL C 16 versus fasted (g)), showing upregulated or downregulated kinase networks. For a-g, n = 4 mice. For d, darker colour intensity reflects higher kinase score. h, Immunoblots (IB) and quantification for indicated proteins in livers of 2-10-month-old male and female mice that were fed or fasted for indicated  Article https://doi.org/10.1038/s41556-023-01163-3 loss of each gene on fasting-induced increases in liver triglycerides. While control and mTORC1 inactivated (Raptor KO ) livers showed equivalent liver triglycerides during fasting, hyperactivation of mTORC1 (Tsc1 KO ) lowered liver triglycerides (Fig. 2c) consistent with the role of mTORC1 in VLDL secretion 24 . Surprisingly, in contrast to the established triglyceride lowering effect of Rictor loss in fed/obesogenic states 25    Article https://doi.org/10.1038/s41556-023-01163-3 inactivating mTORC2 (Rictor KO ) markedly increased liver triglycerides and lipid droplet content during fasting (Fig. 2d,e) without affecting circulating FFA (Fig. 2f). Interestingly, Rictor KO livers displayed lower oxygen consumption rates (OCRs) (Fig. 2g) and accumulation of substrates for mitochondrial respiration, acyl carnitines ( Fig. 2h), which in conjunction with reduced mitochondrial membrane potential in siRictor cells (Fig. 2i) indicate mitochondrial insufficiency. Decreased OCR was not due to impaired expression of FFA oxidation or electron transport genes. In fact, fasted Rictor KO livers displayed increased expression of genes involved in fat oxidation (Ppara, Cpt1a, Cpt1b, Cact and Cpt2), electron transport (Cox4, Nd1 and Cytb) and mitochondrial biogenesis (Ppargc1a) (Extended Data Fig. 3d-s). Levels of mitochondrial fatty acid uptake proteins (CPT1A/CPT2/CACT) and electron transport chain components, NDUFB8 (complex I), SDHB (II), UQCRC2 (III), MT-CO1 (IV) and ATP5A (V) were also comparable in control and Rictor KO livers (Extended Data Fig. 3t,u). Interestingly, loss of Rictor led to increased expression of mitochondrial fusion genes Mfn2 and Opa1 during fasting without affecting fission genes Mff and Dnm1l (DRP1) (Extended Data Fig. 3v-z). Consistent with mitochondrial insufficiency, 3D transmission electron microscopy (TEM) revealed mitochondrial distention and blunted networking ( Fig. 2j and Supplementary Videos 1 and 2) suggesting that reactivation of mTORC2 during fasting supports mitochondrial dynamics.

Loss of mTORC2 blocks fasting-induced mitochondrial fission
To determine whether mTORC2 regulates mitochondrial dynamics during fasting, we first determined how fasting impacts mitochondrial dynamics. TEM of fasted livers revealed increased mitochondrial number and higher frequency of mitochondria with reduced area, perimeter and length (Extended Data Fig. 4a- Since perturbations in membrane lipids or ER stress could alter mitochondrial dynamics, we tested if these changes associate with impaired fission in our model. Loss of Rictor mildly affected lipid composition of MAMs (Supplementary Table 3) without inducing ER stress or proteostasis failure (Extended Data Fig. 5e), excluding their contribution to impaired fission. Because mTORC2 localizes to MAMs to regulate Ca 2+ homeostasis and apoptosis 28 , we envisioned that mTORC2 at MAMs also regulates fission. Indeed, MAMs from 14-16 h fasted control livers revealed the presence of RICTOR, fission proteins 4 MFF and DRP1, and fusion proteins OPA1 (ref. 29), MFN1 and MFN2 (Fig. 3c). By contrast, MAMs from fasted Rictor KO livers showed markedly reduced MFF levels without affecting P-DRP1 Ser616 , P-DRP1 Ser637 , DRP1, MFN1, MFN2 or OPA1 levels ( Fig. 3c) supporting that loss of mTORC2 impairs mitochondrial fission.

mTORC2-SGK1 phosphorylates NDRG1 at Ser336
To determine whether phosphorylated targets of mTORC2 (ref. 32) support fission, we used quantitative nano liquid chromatography coupled online with tandem mass spectrometry (nLC-MS/MS) in control and Rictor KO livers (Fig. 4a), which identified 4,553 phosphosites from 1,712 phosphoproteins. Of these, 309 phosphosites (145 upregulated and 164 downregulated) (6.79%) on 212 phosphoproteins (12.38%) were significantly modulated in Rictor KO livers (Extended Data Fig. 6a and Supplementary Table 4). Gene Ontology and enrichment map network analysis 33 revealed that the hypophosphorylated clusters in Rictor KO livers were related to cytoskeleton and cellular architecture, mRNA processing and splicing, protein targeting and regulation of cellular catabolic processes ( Fig. 4b and Supplementary Tables 2 and 5). The denser cluster populated by both upregulated and downregu lated phosphoproteins contained the term 'regulation of metabolism'. Since protein function is modulated by site-specific phosphorylation or cumulative phosphorylation of multiple phosphosites 34 , we measured the overall phosphorylation status (∆Ps) of Quantification for mitochondrial number is shown. b, TEM in fed or 14-16 h fasted Con and Rictor KO livers of 4-9-month-old male mice. Fed Con (n = 7), fasted Con (n = 9) and fed or fasted Rictor KO mice (n = 5). Quantification for percentage of mitochondria-ER contacts is shown. Red arrowheads depict contact sites. c, Immunoblots and quantification of indicated proteins in homogenates (Hom), pure mitochondria (Mp), MAMs, cytosol (Cyt) and ER fractions from 14-16 h fasted Con (n = 3) and Rictor KO livers (n = 5). Two livers were pooled to generate one sample. d, Live cell imaging and quantification for fission and fusion rates in siCon, siRictor or siDnm1l NIH3T3 cells cultured in serum-free medium for 30 min in presence of MitoTracker green to stain for mitochondria (siCon 12 cells, siRictor 11 cells (fission rate) and n = 12 cells (fusion rate), and siDnm1l 11 cells from n = 8 independent experiments; each cell was tracked on an independent plate). White arrowheads depict mitochondrial constriction sites. Yellow arrowheads depict daughter mitochondria arising from fission at a mitochondrial constriction. Scale bar, 2 µm. Please refer to Supplementary Videos 3 (siCon cells), 4 (siRictor cells) and 5 (siDnm1l cells). e, Representative IB for indicated proteins in siCon, siDnm1l and siRictor NIH3T3 cells. Blots are representative of n = 8 (DRP1) and n = 4 (RICTOR) independent experiments obtaining similar results. f, Representative confocal images of (top) AML12 and (bottom) HepG2 cells knocked down for Rictor, and corresponding controls in serum-free medium for 30 min in presence of MitoTracker green to stain for mitochondria. Magnified insets are shown. Quantifications for mitochondrial number and mitochondrial size/shape descriptors (area, perimeter and circularity) are shown (AML12 45 siCon or siRictor cells; HepG2 35 siCon and 38 siRICTOR cells from n = 3 independent experiments each). Ponceau is loading control. Individual replicates and means are shown. *P < 0.05, **P < 0.01 and ****P < 0.0001, one-way ANOVA followed by Tukey's multiple comparisons test (a and d); two-way ANOVA followed by Tukey's multiple comparisons test (b); two-tailed unpaired Student's t-test (c and f). Please refer to Supplementary Article https://doi.org/10.1038/s41556-023-01163-3 phosphoproteins in our dataset, which revealed hyperphosphorylation (∆Ps > 2σ) in 37 phosphoproteins and hypophosphorylation (∆Ps < −2σ) in 49 phosphoproteins (Fig. 4c). Interestingly, phosphorylation of mitophagy receptor BNIP3 at Ser79 and Ser88 was significantly reduced in Rictor KO livers ( Fig. 4d and Extended Data Fig. 6b) with no known roles assigned to BNIP3 Ser79/Ser88 . We also focused on NDRG1 (   Supplementary Table 6). Since NDRG1 is present in MAMs (Extended Data Fig. 6g), we sought to confirm that mTORC2-SGK1 indeed phosphorylates NDRG1 at Ser336. Accordingly, phosphoproteomics and relative quantification of extracted ion chromatogram of peptide SRTASGSSVTS(p)LEGTRSR, corresponding to Flag-NDRG1, from siCon or siRictor cells (Extended Data Fig. 6h-j and Supplementary Table 7) revealed reduced enrichment in siRictor cells compared with siCon cells, confirming that mTORC2 phosphorylates NDRG1 at Ser336.

NDRG1 Ser336Ala mutant livers exhibit fission failure
To determine whether NDRG1 Ser336Ala mutant livers recapitulate the mitochondrial phenotype of Rictor KO livers, we expressed Flag-NDRG1 WT or Flag-NDRG1 Ser336Ala in livers silenced for endogenous Ndrg1 and confirmed equivalent Flag expression by immunohistochemistry (Extended Data Fig. 8d). Consistent with our observations in Rictor KO livers, fasted NDRG1 Ser336Ala livers showed enlarged mitochondria with reduced mitochondrial number, and increased area and perimeter when compared with NDRG1 WT and untransfected livers (Con) (Fig. 4g), reflecting impaired fission. As observed in Rictor KO livers, fasted NDRG1 Ser336Ala livers showed reduced cellular respiration (Fig. 4h). Furthermore, when compared with corresponding controls, MAMs from Rictor KO (Fig. 3c) and NDRG1 Ser336Ala livers ( Fig. 4i), each showed lower levels of MFF without affecting levels of total and phosphorylated DRP1 Ser616 and DRP1 Ser637 , which modulate dynamics 31 (Fig. 3c). Hence, our data support a role for the mTORC2-NDRG1 Ser336 axis in driving mitochondrial fission.

NDRG1 requires MFF, but not DRP1, for mitochondrial fission
To determine how NDRG1 facilitates fission, we used time-lapse microscopy to test if NDRG1 WT interacts with mitochondria. Interestingly, NDRG1 WT frequently co-localized with a constricted region of mitochondria, culminating in fission (Fig. 5a-   8) but exhibited extended futile interactions (~289.8 ± 26.7 s) with mitochondria that did not lead to fission (Fig. 5a-c and Supplementary Video 9). Consistently, silencing Rictor led to extended futile interactions of NDRG1 WT with mitochondria (409.5 ± 63.7 s) and blocked the ability of NDRG1 WT to divide mitochondria (Fig. 5a-c and Supplementary Video 10), linking mTORC2-driven NDRG1 Ser336 phosphorylation to mitochondrial fission. To determine whether NDRG1-mediated fission requires DRP1, we attempted to KO Dnm1l using CRISPR, but failed to generate viable healthy cells, and therefore this limit in interpretation remains. However, upon using small interfering RNAs (siRNAs) to deplete Dnm1l, mitochondrial division via NDRG1 WT remained intact in siDnm1l cells (Fig. 5a-c and Supplementary Video 11). Indeed, despite >90% loss of Dnm1l, NDRG1 WT continued to engage with mitochondria resulting in fission in ~102 ± 24.6 s. In fact, expressing NDRG1 WT completely restored the altered mitochondrial number, area, perimeter and circularity in siDnm1l cells (Fig. 5d and Extended Data Fig. 9a). By contrast, NDRG1 WT failed to restore the alterations in mitochondrial number, area, perimeter and circularity in siMff cells (Fig. 5e and Extended Data Fig. 9b) suggesting that, although DRP1 is a key regulator of fission, it appears to not influence mitochondrial fission via the mTORC2-NDRG1 axis. By contrast, the mTORC2-NDRG1 axis requires MFF for fission, supported by data showing reduced MFF enrichment in MAMs from Rictor KO (Fig. 3c) and NDRG1 Ser336Ala livers (Fig. 4i) and that siMff cells resist NDRG1 WT -mediated fission (Fig. 5e).

Phosphorylated NDRG1 Ser336 binds CDC42 to drive fission
Since NDRG1 does not exhibit the intrinsic GTPase activity required for membrane scission 38,39 , we asked whether P-NDRG1 Ser336 engages with proteins with intrinsic GTPase activity to facilitate fission. Accordingly, proteomics to identify proteins bound to Flag-tagged NDRG1 WT , but not NDRG1 Ser336Ala , revealed interaction with CDC42, a RHO GTPase that regulates actin cytoskeleton 40 and cytokinesis 9 (Fig. 6a and Supplementary Table 8). Indeed, when compared with NDRG1 WT , NDRG1 Ser336Ala displayed reduced, albeit modest, binding to CDC42, ARHGEF10 (RHO GEF that activates RHO GTPases by stimulating GDP/GTP exchange) 41 , and ARHGAP35 (RHO GAP that facilitates GTP hydrolysis to inactivate RHO GTPases) 41 . Consequently, we hypothesized that GTPase CDC42 mediates the effects of mTORC2-NDRG1 Ser336 on mitochondrial fission. Supporting this hypothesis, co-immunoprecipitation (co-IP) confirmed that exogenously expressed GFP-CDC42 interacts with Flag-NDRG1 WT but fails to interact with mutant NDRG1 Ser336Ala (Fig. 6b). Furthermore, Flag-NDRG1 WT interacts with both mCherry-CDC42 WT and mutant CDC42 Thr17Asn , which is expected to be predominantly GDP-bound (Extended Data Fig. 9c), indicating that CDC42-GTP loading is not required for CDC42-NDRG1 interaction. However, NDRG1 Ser336 phosphorylation and CDC42-GTP loading are each critical for mitochondrial fission. Indeed, silencing Cdc42 (Extended Data Fig. 9d) led to prolonged and futile engagement of NDRG1 WT with mitochondria and blocked fission (Fig. 6c-e and Supplementary Videos 12 and 13). Furthermore, while CDC42 WT engaged with, and divided, mitochondria in ~157 ± 26 s, mutant CDC42 Thr17Asn exhibited prolonged (~339 ± 58 s) and futile interactions with mitochondria (Extended Data Fig. 9e-g and Supplementary Videos 14 and 15). Supporting that the mTORC2-NDRG1-CDC42 axis drives fission, knocking down Rictor or Ndrg1 or Cdc42 each reduced mitochondrial number and circularity, and increased mitochondrial area, perimeter and elongation (Fig. 6f), recapitulating the fission failure phenotype of siMff or siDnm1l cells (Fig. 6f). Consistently, silencing Cdc42 reduced mitochondrial membrane potential (Fig. 6g), suggesting that CDC42 cooperates with P-NDRG1 Ser336 to support mitochondrial division.
To examine if the identified candidates regulate mitochondrial fission, we transfected NIH3T3 cells with siRNAs against each target (Extended Data Fig. 10b), except Cdc42ep2 since silencing it severely reduced viability. Our results indicate that CDC42 and its family of effectors/regulators control mitochondrial fission, since deleting Cdc42 or CDC42 activators, Arhgef10 and Bin3, or CDC42 downstream effectors, Cdc42ep4/BORG4 or Cdc42ep1/BORG5, each resulted in increased mitochondrial area, perimeter and elongation, reflecting impaired fission (Fig. 7c). RHOGDI proteins can act as negative regulators of RHO GTPases by retaining RHO GTPases in the cytosol, inhibiting  -test (a, b, e and g)  their GTPase activity, and preventing their interaction with GEFs, GAPs and effectors 43,44 . Accordingly, we suspect that silencing Arhgdia/ RHOGDI1 releases CDC42 from the inhibitory effect of ARHGDIA/ RHOGDI1, leading to fission. Consistently, silencing Arhgdia/RHOGDI1 (but not Arhgdib/RHOGDI2) increased mitochondrial number (Fig. 7c), reflecting increased fission. Not all RHO GTPases impact mitochondrial dynamics, since depleting the RHO GTPase Rhobtb1 gene had no effect on mitochondrial morphology, suggesting specificity of RHO GTPase CDC42 towards fission. We also found that silencing IQGAP1, which regulates CDC42 as an upstream scaffold and as a downstream effector of CDC42 (ref. 42), increased mitochondrial number (Fig. 7c). Indeed, as a scaffold protein, IQGAP1 provides a molecular link between Ca 2+ /calmodulin and CDC42-mediated processes 45 , while as a downstream effector, CDC42 enhances the F-actin-cross-linking activity of IQGAP1 during actin reorganization 46 . Since ARHGAP35 inactivates GTPases, we anticipated that depleting Arhgap35 would stimulate CDC42, leading to fission; however, knocking down Arhgap35 decreased mitochondrial number, reflecting fission failure (Fig. 7c). This probably reflects the complex regulation of CDC42 requiring subsequent inactivation to complete its function 47,48 , as well as specificity among the different effectors and regulators in stimulating fission. Alternatively, ARHGAP35 is perhaps not active towards CDC42 and affects, instead, an antagonistic GTPase. Consistent with these findings, in addition to CDC42, we detected the presence in MAMs of CDC42 effector, CDC42EP1/BORG5, and ARHGAP35 and ARHGDIA/ RHOGDI1 (Extended Data Fig. 10c,d). Interestingly, levels of ARHGAP35, CDC42EP1/BORG5 and ARHGDIA/RHOGDI1 in MAMs from Rictor KO (Extended Data Fig. 10c) and NDRG1 Ser336Ala expressing livers (Extended Data Fig. 10d) were comparable to those in corresponding controls, indicating that fission is probably regulated at the level of recruitment of CDC42 to MAMs.
Since CDC42 action is regulated by membrane binding 49 and GTP loading 50 , we suspect that mTORC2-driven NDRG1 Ser336 phosphorylation is a signal to recruit CDC42 to MAMs for its activation to drive fission. Indeed, CDC42 and RHOA 51 (Fig. 7d), but not dynamins, were abundantly present in MAMs from fasted livers. By contrast, MAMs from fasted Rictor KO (Fig. 7d) and NDRG1 Ser336Ala livers (Fig. 7e) each showed markedly reduced CDC42 levels without affecting RHOA levels, suggesting that the mTORC2-NDRG1 axis recruits CDC42 to MAMs. Given the enrichment of CDC42 in MAMs in an mTORC2-and NDRG1-sensitive manner, it is likely that CDC42 governs local downstream mechanisms that control fission. Since dynamic cycling of actin through populations of mitochondria controls fission 52,53 , we asked whether CDC42 mediates the effect of mTORC2-NDRG1 on mitochondrial fission by remodelling actin cytoskeleton. Indeed, in control cells, actin assembled around mitochondria to generate ring-like structures consistent with maintained fission 53 (Fig. 7f). By contrast, silencing Cdc42 decreased co-localization of actin with mitochondria, which correlated with elongated mitochondria (Fig. 7f), suggesting that CDC42 facilitates the organization of actin around mitochondria to enable fission.

Discussion
In sum, we show that the typically nutrient-responsive mTORC2 is paradoxically reactivated by fasting to regulate NDRG1 Ser336 phosphorylation, which serves to recruit CDC42 to MAMs to drive mitochondrial fission. In support of this model (Fig. 7g), NDRG1 engages with mitochondrial constrictions to facilitate fission, and that fission events are blocked in cells expressing phosphorylation-deficient NDRG1 Ser336Ala or in cells lacking Rictor or Cdc42 or the identified CDC42 effector/ regulators (Fig. 7g), thus revealing an mTORC2-NDRG1-CDC42 axis facilitating mitochondrial fission during fasting.
Fasting and feeding are hormonally distinct physiological states 19 . While nutrient deprivation in cultured cells blocks mitochondrial fission to preserve ATP synthesis and cell viability 54,55 , cultured cells do not completely recapitulate the complex physiology of intact organisms. In fact, we show that the highly integrated liver exhibits marked increases in fission during an acute fast. Indeed, switching between nutrient availability and deprivation modulates mitochondrial cristae and ER contacts, which per se impact mitochondrial dynamics 56,57 . In keeping with this, we suspect that fasting-induced increases in adipose lipolysis and increased availability of lipids reactivate mTOR during fasting. Indeed, cholesterol 58 and phosphatidic acid 59 activate mTORC1 in vitro, and we show here that exposure to dietary corn oil or fasting each activates mTORC2 in liver, as has been shown for mTORC1 in starved cultured cells 60 . Although no function has been assigned to fasting-induced reactivation of mTOR, we demonstrate that paradoxical reactivation of mTORC2 during fasting is required for mitochondrial remodelling to possibly support the increased energetic demands of fasting. In fact, enzymatic reactions, for example, those part of the Krebs cycle, appear to be sensitive to changes in mitochondrial shape, volume and connectivity 61 . Consistently, not only does loss of Rictor impact mitochondrial fission, we also noted marked accumulation of acylcarnitines, a metabolic signature consisted with dampened mitochondrial respiration.
Mitochondrial division is tightly orchestrated by recruitment of dynamin-related GTPase DRP1 from the cytosol to MAMs by the mitochondrial outer membrane receptor MFF 4 . DRP1 oligomerization and interaction with actin filaments promote scission in a GTP hydrolysis-dependent manner. Indeed, overexpression of MFF fails to restore fission in cells co-expressing the assembly-defective DRP1 mutant, indicating that MFF acts via DRP1 (ref. 4). Yet, our data show that DRP1 is dispensable for mTORC2-NDRG1-mediated mitochondrial fission, since overexpressing NDRG1 WT restores mitochondrial fission in DRP1-deficient cells, and perhaps most surprisingly, NDRG1 WT fails to restore fission when MFF is depleted. These data suggest that N values for number of mice per fraction are indicated in parentheses. CDC42: all fractions from NDRG1 WT and NDRG1 Ser336Ala mice are n = 5 except for NDRG1 Ser336Ala MAMs where n = 4; RHOA: n = 6 mice for all groups. Ponceau is loading control. f, Representative confocal images of siCon or siCdc42 NIH3T3 cells expressing mCherry-Lifeact-7 and cultured in serum-free medium for 30 min in presence of MitoTracker green. Magnified insets are shown. Quantification for percentage co-localization of mCherry-Lifeact-7 with mitochondria is shown (siCon 33 cells and siCdc42 30 cells from n = 5 (siCon) or n = 4 (siCdc42) independent experiments). g, Reactivation of mTORC2 during fasting phosphorylates NDRG1 at Ser336, which engages with mitochondria and recruits CDC42 to mitochondria-ER contact sites wherein CDC42 and its effector proteins orchestrate fission. Individual replicates and means are shown. *P < 0.05 and **P < 0.01, two-tailed unpaired Student's t-test. NS, not significant. Please refer to Supplementary   mTORC2-NDRG1-mediated fission is dependent on MFF but appears to not require DRP1. Supporting this possibility, loss of Rictor or expressing the NDRG1 Ser336Ala mutant each markedly reduced MFF levels in MAMs without affecting DRP1 levels. These findings suggest that roles for MFF in fission are complex and not restricted to merely serving as a receptor for DRP1 recruitment 4 . How then does NDRG1 drive mitochondrial fission? Since NDRG1 lacks GTP hydrolysis activity, a requirement for fission 38,39 , we examined whether NDRG1 engages with additional GTPases to facilitate fission. Here we identify the small GTPase CDC42 as a binding partner of NDRG1 that fails to interact with the phosphorylation-deficient NDRG1 Ser336Ala mutant. Indeed, time-lapse imaging revealed that NDRG1 and CDC42 both engage at mitochondrial constrictions to facilitate fission, and that in absence of Cdc42 or presence of inactive GDP-bound CDC42 Thr17Asn mutant, NDRG1 fails to cut mitochondria. Furthermore, we detected the presence of CDC42 in MAMs, the enrichment of which appears to depend on an intact functional mTORC2-NDRG1 axis since loss of Rictor or expressing the NDRG1 Ser336Ala mutant each markedly reduced CDC42 levels in MAMs. Given that CDC42 modulates the actin cytoskeleton 40 , it is tempting to speculate that recruitment of CDC42 to MAMs by the mTORC2-NDRG1 axis orchestrates a local interplay between actin tubules and ER in driving scission, although careful future assessments are needed to conclusively demonstrate the same. Since Rictor insufficiency shortens lifespan 62,63 , and given the age-related impairment in mitochondrial function, it is also tempting to speculate that stimulation of mTORC2 to sustain mitochondrial fission could potentially delay age-related diseases in which defective mitochondrial dynamics play a part.

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Corn oil
Corn oil (Sigma-Aldrich, 8267) is protein free, and provides fatty acids and monoacylglycerols, which are absorbed by the gut and delivered systemically. The composition of corn oil is detailed in Supplementary  Table 11.

In vitro transfections of nucleic acids
In vitro transfections were performed using Lipofectamine 3000 (Invitrogen, L3000). For expression of DNA plasmids, 120,000 NIH3T3 cells ml −1 of growth medium were transfected with 1 µg of DNA and plated in 12-well plate dishes for 48 h. For gene silencing, 120,000 cells ml −1 of growth medium were transfected with siRNA for 48 h (Supplementary Table 12). Scrambled RNA was used as negative control (siCon). Silencing efficiency was confirmed by western blotting or qPCR.

In vivo delivery of nucleic acids
In vivo delivery of plasmid DNAs was performed via in vivo-jetPEI (Polyplus-transfection SA, 201-50G) as per the manufacturer's instructions. Briefly, 100 µg of NDRG1 WT or NDRG1 Ser336Ala _pcDNA3.1+/ C-(K)-DYK was diluted in glucose solution and combined with 7 µl of in vivo-jetPEI for 15 min at room temperature. Then 200 µl of transfection mix was administered retro-orbitally to C57BL/6 mice in a single injection 24 h before tissue collection. Transfection efficiency was determined by immunohistochemistry. Livers from non-transfected mice were used as negative controls. In vivo siRNA delivery was performed using Invivofectamine 3.0 Reagent (Invitrogen, IVF3005) as per the manufacturer's instructions. Briefly, 50 µg of siRNAs was mixed with complexation buffer, added to Invivofectamine 3.0 Reagent (1:1 ratio) and incubated for 30 min at 50 °C. The mix was diluted in PBS (pH 7.4), and 200 µl of siRNA mix was administered retro-orbitally to C57BL/6 mice every 24 h for 3 consecutive days before tissue collection.

RNA isolation and real-time PCR
mRNA expression was performed as described 66 using M-MLV Reverse Transcriptase (Invitrogen, 28025). The primers are detailed in Supplementary Table 13.

Western blotting
Total cell lysates from cells in culture were prepared using lysis buffer (20 mM Tris pH 7.5, 50 mM NaCl, 0.5%, 1 mM EDTA, 1 mM EGTA and 1% Triton X-100) supplemented with complete EDTA-free protease inhibitor (Roche, 11873580001) and phosphatase inhibitor cocktails 2 and 3 (Sigma-Aldrich, P5726 and P0044). Total protein from liver or epididymal adipose tissue was isolated in RIPA buffer (50 mM Tris pH 8.0, 150 mM NaCl, 0.5% sodium deoxycholate, 1% SDS and 1% NP-40) supplemented with protease/phosphatase inhibitors. Total protein from soleus muscle was isolated as described 67 . Lysates were centrifuged at 17,000g for 30 min at 4 °C, and supernatants were immunoblotted by denaturing 20-30 µg of protein at 95 °C for 5 min in 3× Laemmli buffer. For analysis of OXPHOS, samples were boiled at 50 °C for 5 min and resolved by SDS-PAGE as described 68 . Protein bands were normalized to Ponceau S and quantified by ImageJ (National Institutes of Health, NIH). Antibodies are detailed in Supplementary Table 14.

Subcellular fractionation
Fresh livers were fractionated for isolation of MAMs, pure mitochondria, cytosol and ER fractions as described 69 . Cytochrome c (CYT c) and Voltage Dependent Anion Channel 1 (VDAC1) were used as enrichment/ purity markers for mitochondria, long-chain fatty acid coenzyme A ligase 4 (FACL4) as marker for MAM, calreticulin as marker for MAMs and ER, and tubulin as marker for cytoplasm.

Sample preparation for phosphoproteomics
Liver (500 µg), liver MAM fractions (700 µg) or co-IP eluents from Flag pulldowns performed in total cell lysates (700 µg) of siCon or siRictor NIH3T3 cells co-expressing Flag-NDRG1 WT were homogenized in 2% SDS/5 mM dithiothreitol (with protease/phosphatase inhibitors) to retrieve proteins in solution and incubated for 1 h at room temperature for disulfide bond reduction. Proteins were alkylated using 20 mM iodoacetamide for 30 min in the dark. Protein digestion was performed utilizing S-trap mini cartridges (ProtiFi) as per the manufacturer's instructions. Phosphorylated peptides were enriched from the S-trap eluate using titanium dioxide beads (TiO 2 , GL Sciences) as described 70 . Following TiO 2 enrichment, peptides were concentrated with a speed vac, desalted in HLB resin (Waters) and concentrated in a speed vac once more before analysing peptides by nLC-MS/MS.

nLC-MS/MS acquisition
Samples were resuspended in 10 µl of water/0.1% trifluoroacetic acid and loaded onto a Dionex RSLC Ultimate 300 (Thermo Scientific) coupled online with an Orbitrap Fusion Lumos (Thermo Scientific). The two-column chromatographic separation system consisted of a C18 trap cartridge (300 µm internal diameter (ID), 5 mm length) and a picofrit analytical column (75 µm ID, 30 cm length) packed in-house with reversed-phase Repro-Sil Pur C18-AQ 3 µm resin. Peptides were separated using a 180 min gradient from 2% to 28% buffer B (buffer A: 0.1% formic acid; buffer B: 80% acetonitrile/0.1% formic acid) at a flow rate of 300 nl min −1 . The mass spectrometer acquired spectra in a data-dependent acquisition mode. Briefly, the full MS scan was set to 300-1,200 m/z in the Orbitrap with a resolution of 120,000 (at 200 m/z) and an AGC target of 5 × 10 5 . MS/MS was performed in the ion trap using top speed mode (2 s), an AGC target of 10 × 10 4 and higher collisional dissociation (HCD) collision energy of 30. Two additional targeted scans were added in each instrument duty cycle to detect the low-abundance NDRG1 Ser336 peptide: a selected ion monitoring scan for the intact mass quantification and a targeted MS/MS scan for identification of the peptide.
Phosphoproteomics data analysis was conducted using Proteome Discoverer v2.4 (Thermo Scientific) at standard settings for tolerances, modifications and filters, and phosphorylation on Ser/Thr/tyrosine as dynamic modifications. SwissProt mouse proteome database was used (downloaded August 2019). Peptide abundance was obtained using the intensity of the extracted ion chromatogram; values were log 2 transformed and normalized, and missing values were imputed as described 71 . Comparisons between groups were performed in a binary manner; each sample type was compared with the fasted condition utilizing a two-tails heteroscedastic t-test (significant, if P value < 0.05). The data distribution was assumed to be normal.
Significantly modified proteins were selected by Benjamini-Hochberg correction (P < 0.05). When false discovery rate correction led to no hit, inspection of uncorrected P value distribution was performed: if an anti-conservative distribution was observed, we applied an alternative method of false discovery rate control by combining threshold for significance (P < 0.05) with fold-change cut-off (fold-change >1.5) as suggested 72 . Phosphorylation state change (∆Ps) for individual proteins was calculated as described 34 , as the sum of log 2 (fold change) value of all phosphopeptides with statistically significant changes (P < 0.05) compared to control. If none of the phosphopeptide P values is below 0.05, the ∆Ps value will be zero. We applied a stringent cut-off for ∆Ps value at two standard deviations (2σ) to represent the concept of cumulative phosphorylation. Gene Ontology was performed using BINGO or Enrichr 73 . In the enrichment map-based network visualization of Gene Ontology enrichment of differentially modulated phosphosites, blue edges show similarity between decreased phosphosites while red nodes show similarity between increased phosphosites; node size indicates the number of proteins per node; major clusters are circled, and the associated names represent major functional associations. The enrichment map was generated in Cytoscape Phosphosites showing significant regulation between groups were used to predict the kinase responsible for their catalysis using the iGPS software 12 . Significantly regulated phosphorylation events were used to predict the kinases responsible for their catalysis using iGPS 12 . Positive kinase scores represent most confident and frequent predictions for upregulated phosphosites, with blue representing downregulated phosphosites. The higher the cumulative score retrieved from iGPS, the more intense the colour coding of the bubbles in the network. Upregulated and downregulated refers to numerator and denominator as defined in the header of each panel. The bubble size is scaled on the basis of the number of phosphorylation events predicted to be catalysed by the given kinase. The connector lines represent previously associated genetic interactions between listed proteins retrieved from the database STRING v. 11 (ref. 76). The network was displayed using Cytoscape 77 .
Seahorse XF Cell Mito Stress Test was performed as per the manufacturer's instructions (Agilent Technologies). Briefly, 12,000 NIH3T3 cells per 200 µl of medium were transfected with DNAs and/or siRNAs and seeded onto a Seahorse XF96 Cell Culture Microplate (Agilent Technologies, 101085-004) for 32 h. After 16 h of stress in low-glucose medium (1 g l −1 ; Agilent Technologies, 103577), cells were washed with PBS, cultured in 165 µl of XF Base Medium (Agilent Technologies, 103335) supplemented with 1 g l −1 d-glucose, 2 mM sodium pyruvate (Gibco, 11360) and 4 mM l-glutamine (Gibco, 25030) and incubated at 37 °C without CO 2 for 1 h. OA (0.25 mM) was added, and the microplate was loaded into XF Analyzer. Basal OCR measurements were recorded four times (mix 3 min, wait 2 min, measure 3 min) after sequential injections of oligomycin (1 µM), carbonyl cyanide-4 (trifluoromethoxy) phenylhydrazone (FCCP; 20 µM) and rotenone/antimycin (1 µM) with four readings (mix 3 min, wait 2 min, measure 3 min) after each injection. OCR was normalized to cell number estimated with CyQUANT Cell Proliferation Assay (Invitrogen, C7026) as per the manufacturer's instructions. Mitochondrial parameters were calculated as per the manufacturer's instructions. OCRs of liver explants were performed as described 66 .

Histological analyses
Flag was detected using a Mouse on Mouse (MOM) ImmPRESS HRP (Peroxidase) Polymer Kit (Vector Laboratories, MP-2400). Paraffin-embedded livers were cut into 5 µm sections, deparaffinated in xylene and rehydrated in a series of graded alcohols and water. For antigen unmasking, sections were incubated in citrate-based antigen unmasking solution (pH 6.0; Vector Laboratories, H-3300) at high temperature for 20 min. After blocking in BLOXALL Endogenous Blocking Solution (Vector Laboratories, SP-6000) for 10 min and a 1 h incubation in MOM Mouse IgG Blocking Reagent, sections were stained with mouse monoclonal anti-DYKDDDDK Tag antibody (1:100; Cell Signaling Technology, 8146) in 2.5% normal horse serum MOM solution overnight at 4 °C. DYKDDDDK signal was revealed by incubation with MOM ImmPRESS Reagent for 10 min and enhanced with ImmPACT DAB EqV Peroxidase (HRP) Substrate (Vector laboratories, SK-4103) for 1 min. Sections were counterstained with haematoxylin, dehydrated, mounted with Permount mounting medium (Fisher, SP15) and imaged in a Zeiss Axiolab 5 microscope/Axiocam 305 colour camera (Carl Zeiss Microscopy). Quantification of Flag percentage area was performed as described 78 .

Oil Red O staining
Oil Red O staining was performed as described 79 .

Confocal microscopy
Was performed as described 68 . For Flag detection, DYKDDDDK Tag Rabbit antibody was used at 1:100 dilution (Cell Signaling Technology, 14793). Where indicated, 30 min before fixation with 4% paraformaldehyde, cells were incubated with 100 nM MitoTracker Red CMXRos (Invitrogen, M7512) to assess mitochondrial membrane potential. Mounted coverslips were imaged on a Leica TCS SP8 Confocal Laser Scanning Microscope (Leica Microsystems) with ×63 objective and 1.4 numerical aperture. Quantification of MitoTracker Red CMXRos fluorescence intensity per cell was performed using ImageJ (NIH) and expressed as mean integrated density. For detection of BODIPY FL C 16 in vivo, sections from freshly isolated livers were mounted with Fluoromount-G medium (SouthernBiotech, 0100) and imaged on Leica TCS SP8 Confocal Laser Scanning Microscope with ×10 objective and 1.4 numerical aperture.

Live cell imaging
Cells were transfected with siRNA and/or DNA as above and seeded onto a glass-bottom 35 mm culture dish (MatTek Corporation, P35G-1.5-14-C) for 48 h. After PBS washing, cells were incubated in serum-free DMEM in presence of MitoTracker Green FM (500 nM; Invitrogen, M7514) or ER-Tracker Green (500 nM; Invitrogen, E34251) for 30 min to stain mitochondria and ER, respectively. Cells were washed once with PBS and incubated in red phenol-free DMEM (Gibco, 31053) with 4 mM l-glutamine and 12 mM HEPES (pH 7.4), and imaged using a Leica TCS SP8 confocal laser scanning microscope (Leica Microsystems) and single planes were acquired with ×63 objective and 1.4 numerical aperture. For time-lapse imaging, cells were tracked at a rate of one frame per 13 or 27 s (for single or simultaneous dual-channel acquisition, respectively) over 10 min.
Image analysis was done with ImageJ (NIH). Individual frames were denoised by applying Gaussian filter and a region of interest (ROI) of 35 µm 2 was selected across the different experimental conditions. After image auto-thresholding, quantification of mitochondrial number and morphology parameters was performed using the 'analyze particles' macro as described 80 . Mitochondrial elongation was calculated as the inverse of circularity 81 . Mitochondrial fission and fusion frequency were calculated as described 82 and expressed as number of events per cell per second. Percentage co-localization was calculated using the JACoP plugin as described 68 .

TEM
Freshly isolated livers were fixed with 2% paraformaldehyde and 2.5% glutaraldehyde in 0.1 M sodium cacodylate, post-fixed with 2% osmium tetroxide, 1.5% potassium ferrocyanide, 0.15 M sodium cacodylate, 2 mM CaCl 2 , followed by 1% thiocarbohydrazide, and then 2% osmium tetroxide, en bloc stained with 1% uranyl acetate and further stained with lead aspartate. Samples were dehydrated through graded series of ethanol and embedded in LX112 resin (LADD Research Industries). Ultrathin (55 nm) sections were cut on a Leica ARTOS 3D ultra microtome and collected onto silicon wafers. Sections were examined on Zeiss Supra 40 Field Emission Scanning Electron Microscope (Carl Zeiss Microscopy, LLC North America) in backscatter mode using an accelerating voltage of 8.0 kV. The number of mitochondria was counted manually in an ROI of 71.2 µm 2 . Quantification of mitochondrial shape descriptors was performed by manual tracing of individual mitochondria using freehand tool. Contact sites between mitochondria and ER (defined to be at 10-30 nm distance 83 ) were quantified, normalized to total number of mitochondria and expressed as percentage. For 3D reconstruction, regions of interest were collected using ATLAS 5.0, with a pixel size of 6.0 and dwell time of 6 µs. Stacks were aligned, and segmentation was done using IMOD 84 . Tomographic reconstruction was performed as described 85 .

Lipidomic analyses
Lipid extracts from liver homogenates, MAM, pure mitochondria and ER fractions were prepared using modified Bligh and Dyer method, spiked with appropriate internal standards, and analysed on an Agilent 1260 Infinity HPLC integrated to Agilent 6490 A QQQ mass spectrometer controlled by Masshunter v 7.0 (Agilent Technologies). Glycerophospholipids and sphingolipids were separated with normal-phase HPLC as described 86 , with a few modifications. An Agilent Zorbax Rx-Sil column (2.1 × 100 mm, 1.8 µm) at 25 °C was used under the following conditions: mobile phase A (chloroform:methanol:ammonium hydroxide, 89.9:10:0.1, v/v) and mobile phase B (chloroform:methanol:water :ammonium hydroxide, 55:39:5.9:0.1, v/v); 95% A for 2 min, decreased linearly to 30% A over 18 min and further decreased to 25% A over 3 min, before returning to 95% over 2 min and held for 6 min. Separation of sterols and glycerolipids was carried out on a reverse phase Agilent Zorbax Eclipse XDB-C18 column (4.6 × 100 mm, 3.5 µm) using an isocratic mobile phase, chloroform:methanol:0.1 M ammonium acetate (25:25:1) at a flow rate of 300 µl min −1 .
Quantification of lipid species was accomplished using multiple reaction monitoring transitions 86 Table 3).

Illustration
The proposed model in Fig. 7g was created with BioRender (BioRender. com).

Statistics
All data are mean of a minimum of three independent experiments unless otherwise stated. Statistical significance was assessed by two-tailed unpaired Student's t-test, one-way or two-way analyses of variance (ANOVAs) followed by Tukey's, Šídák's or Dunnett's multiple-comparisons test. n numbers indicate biological replicates. Statistical summary is presented in Supplementary Table 10. Raw source data are presented in Source Data Extended Data Table 1.

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

Data availability
The mass spectrometry proteomics data have been deposited to the ProteomeXchange Consortium via the PRIDE 88 partner repository, and data are available via ProteomeXchange with identifier PXD041696. Data from this study are available at https://doi.org/10.6084/ m9.figshare.22670575. Source data have been provided with this paper. All other data supporting the findings of this study are available from the corresponding author on reasonable request. , BODIPY FL C 16 (n = 9), refed (n = 4); P-AKT Ser473 /AKT: fasted (n = 17), corn oil (n = 13), BODIPY FL C 16 (n = 9), refed (n = 4); P-AKT Thr308 /AKT: fasted (n = 4), corn oil (n = 3), BODIPY FL C 16 (n = 4), refed (n = 4). Ponceau is the loading control. (g) Circulating free fatty acids (FFA) in 2-7 mo-old male mice fasted for: 0 h (n = 20 mice), 3 h (n = 9 mice), 14 h (n = 3 mice) or 20 h (n = 15 mice). Individual replicates and mean values are shown. *P < 0.05, ***P < 0.001 and ****P < 0.0001, One-way ANOVA followed by Šídák's multiple comparisons test (f); One-way ANOVA followed by Tukey's multiple comparisons test (g). Please refer to Supplementary  Table 10_statistical summary, and Supplementary Tables 1, 2 Representative IB to validate deletion of the indicated genes in livers of 4-6 moold Con, Tsc1 KO or Raptor KO male and female mice. Blots for TSC1 and RAPTOR are representative of n = 7 Con or corresponding KO mice obtaining similar results. (c) IB for mTORC2 signaling and quantification of RICTOR protein levels in liver (n = 5 mice), epididymal white adipose tissue (eWAT) (n = 5 Con and n = 4 Rictor KO mice) and soleus (n = 5 mice) of 4-6 mo-old Con and Rictor KO male mice fasted for 14-16 h. (d-s and v-z) Relative mRNA expression of indicated genes in livers of 4-6 mo-old Con and Rictor KO male mice that were fed or fasted for 14-16 h (n = 7 fed Con or Rictor KO mice, and n = 5 fasted Con or Rictor KO mice). (t) Representative IB for proteins involved in mitochondrial fatty acid uptake, and (u) OXPHOS in whole homogenates (Hom) and pure mitochondrial (Mp) fractions from livers of 5-6 mo-old Con and Rictor KO male mice after 14-16 h fasting (n = 3 Con and n = 5 Rictor KO mice). Ponceau is the loading control. Individual replicates and mean values are shown. *P < 0.05, **P < 0.01, ***P < 0.001 and ****P < 0.0001, twotailed unpaired Student's t-test (c); 2-way ANOVA followed by Tukey's multiple comparisons (d-s and v-z). ns=not significant. Please refer to Supplementary  Table 10_statistical summary. Source numerical data are available in SourceData_ Table 1, and unprocessed blots are available in Source Data Extended Data Fig. 3.