Title: <scp>mTORC</scp> 2 sustains thermogenesis via Akt‐induced glucose uptake and glycolysis in brown adipose tissue
Abstract: Research Article15 January 2016Open Access Transparent process mTORC2 sustains thermogenesis via Akt-induced glucose uptake and glycolysis in brown adipose tissue Verena Albert Verena Albert Biozentrum, University of Basel, Basel, Switzerland Search for more papers by this author Kristoffer Svensson Kristoffer Svensson Biozentrum, University of Basel, Basel, Switzerland Search for more papers by this author Mitsugu Shimobayashi Mitsugu Shimobayashi Biozentrum, University of Basel, Basel, Switzerland Search for more papers by this author Marco Colombi Marco Colombi Biozentrum, University of Basel, Basel, Switzerland Search for more papers by this author Sergio Muñoz Sergio Muñoz Center of Animal Biotechnology and Gene Therapy and Department of Biochemistry and Molecular Biology, School of Veterinary Medicine, Universitat Autònoma de Barcelona, Bellaterra, Spain Centro de Investigación Biomédica en Red de Diabetes y Enfermedades Metabólicas Asociadas (CIBERDEM), Madrid, Spain Search for more papers by this author Veronica Jimenez Veronica Jimenez Center of Animal Biotechnology and Gene Therapy and Department of Biochemistry and Molecular Biology, School of Veterinary Medicine, Universitat Autònoma de Barcelona, Bellaterra, Spain Centro de Investigación Biomédica en Red de Diabetes y Enfermedades Metabólicas Asociadas (CIBERDEM), Madrid, Spain Search for more papers by this author Christoph Handschin Christoph Handschin Biozentrum, University of Basel, Basel, Switzerland Search for more papers by this author Fatima Bosch Fatima Bosch Center of Animal Biotechnology and Gene Therapy and Department of Biochemistry and Molecular Biology, School of Veterinary Medicine, Universitat Autònoma de Barcelona, Bellaterra, Spain Centro de Investigación Biomédica en Red de Diabetes y Enfermedades Metabólicas Asociadas (CIBERDEM), Madrid, Spain Search for more papers by this author Michael N Hall Corresponding Author Michael N Hall Biozentrum, University of Basel, Basel, Switzerland Search for more papers by this author Verena Albert Verena Albert Biozentrum, University of Basel, Basel, Switzerland Search for more papers by this author Kristoffer Svensson Kristoffer Svensson Biozentrum, University of Basel, Basel, Switzerland Search for more papers by this author Mitsugu Shimobayashi Mitsugu Shimobayashi Biozentrum, University of Basel, Basel, Switzerland Search for more papers by this author Marco Colombi Marco Colombi Biozentrum, University of Basel, Basel, Switzerland Search for more papers by this author Sergio Muñoz Sergio Muñoz Center of Animal Biotechnology and Gene Therapy and Department of Biochemistry and Molecular Biology, School of Veterinary Medicine, Universitat Autònoma de Barcelona, Bellaterra, Spain Centro de Investigación Biomédica en Red de Diabetes y Enfermedades Metabólicas Asociadas (CIBERDEM), Madrid, Spain Search for more papers by this author Veronica Jimenez Veronica Jimenez Center of Animal Biotechnology and Gene Therapy and Department of Biochemistry and Molecular Biology, School of Veterinary Medicine, Universitat Autònoma de Barcelona, Bellaterra, Spain Centro de Investigación Biomédica en Red de Diabetes y Enfermedades Metabólicas Asociadas (CIBERDEM), Madrid, Spain Search for more papers by this author Christoph Handschin Christoph Handschin Biozentrum, University of Basel, Basel, Switzerland Search for more papers by this author Fatima Bosch Fatima Bosch Center of Animal Biotechnology and Gene Therapy and Department of Biochemistry and Molecular Biology, School of Veterinary Medicine, Universitat Autònoma de Barcelona, Bellaterra, Spain Centro de Investigación Biomédica en Red de Diabetes y Enfermedades Metabólicas Asociadas (CIBERDEM), Madrid, Spain Search for more papers by this author Michael N Hall Corresponding Author Michael N Hall Biozentrum, University of Basel, Basel, Switzerland Search for more papers by this author Author Information Verena Albert1, Kristoffer Svensson1, Mitsugu Shimobayashi1, Marco Colombi1, Sergio Muñoz2,3, Veronica Jimenez2,3, Christoph Handschin1, Fatima Bosch2,3 and Michael N Hall 1 1Biozentrum, University of Basel, Basel, Switzerland 2Center of Animal Biotechnology and Gene Therapy and Department of Biochemistry and Molecular Biology, School of Veterinary Medicine, Universitat Autònoma de Barcelona, Bellaterra, Spain 3Centro de Investigación Biomédica en Red de Diabetes y Enfermedades Metabólicas Asociadas (CIBERDEM), Madrid, Spain *Corresponding author. Tel: +41 61 267 21 50; E-mail: [email protected] EMBO Mol Med (2016)8:232-246https://doi.org/10.15252/emmm.201505610 PDFDownload PDF of article text and main figures. Peer ReviewDownload a summary of the editorial decision process including editorial decision letters, reviewer comments and author responses to feedback. ToolsAdd to favoritesDownload CitationsTrack CitationsPermissions ShareFacebookTwitterLinked InMendeleyWechatReddit Figures & Info Abstract Activation of non-shivering thermogenesis (NST) in brown adipose tissue (BAT) has been proposed as an anti-obesity treatment. Moreover, cold-induced glucose uptake could normalize blood glucose levels in insulin-resistant patients. It is therefore important to identify novel regulators of NST and cold-induced glucose uptake. Mammalian target of rapamycin complex 2 (mTORC2) mediates insulin-stimulated glucose uptake in metabolic tissues, but its role in NST is unknown. We show that mTORC2 is activated in brown adipocytes upon β-adrenergic stimulation. Furthermore, mice lacking mTORC2 specifically in adipose tissue (AdRiKO mice) are hypothermic, display increased sensitivity to cold, and show impaired cold-induced glucose uptake and glycolysis. Restoration of glucose uptake in BAT by overexpression of hexokinase II or activated Akt2 was sufficient to increase body temperature and improve cold tolerance in AdRiKO mice. Thus, mTORC2 in BAT mediates temperature homeostasis via regulation of cold-induced glucose uptake. Our findings demonstrate the importance of glucose metabolism in temperature regulation. Synopsis Albert et al show that β-adrenergic stimulation activates mTORC2 in brown adipocytes. Active mTORC2 signaling in BAT is required for cold-induced stimulation of glucose uptake and glycolysis to maintain temperature homeostasis upon cold stress. β-adrenergic stimulation activates mTORC2 in brown adipocytes. Mice deficient for mTORC2 in adipose tissue are hypothermic and sensitive to cold. mTORC2 in BAT stimulates cold-induced glucose uptake and glycolysis via Akt. Restoration of glucose uptake in BAT of AdRiKO mice restores temperature homeostasis. Introduction Non-shivering thermogenesis (NST) in brown adipose tissue (BAT) allows mammals to maintain stable body temperature in a cold environment. Upon cold exposure, norepinephrine (NE) is released from sympathetic nerves and binds to adrenergic receptors on brown adipocytes to induce NST. Adrenergic receptor stimulation induces cAMP production and subsequent induction of lipolysis, β-oxidation, and mitochondrial uncoupling (Cannon & Nedergaard, 2004). Mitochondrial uncoupling occurs through activation of uncoupling protein 1 (UCP1). UCP1 is a mitochondrial transmembrane protein specifically expressed in brown adipocytes and brown-like, beige adipocytes. Once activated, UCP1 dissipates the proton gradient across the inner mitochondrial membrane generated by the electron transport chain. This uncouples proton flux into the mitochondria from ATP production, resulting in heat generation (Klaus et al, 1991; Busiello et al, 2015). To compensate for the loss of mitochondrial ATP production due to uncoupling, adrenergic stimulation enhances glucose uptake and glycolysis in BAT (Greco-Perotto et al, 1987; Vallerand et al, 1990; Hao et al, 2015). Due to the ability of BAT to burn energy efficiently and to reduce blood glucose levels, activation of NST has been proposed as an alternative strategy for weight loss in obese patients (Cypess et al, 2009; van Marken Lichtenbelt et al, 2009; Virtanen et al, 2009; Clapham & Arch, 2011) and for normalization of blood glucose levels in insulin-resistant diabetic patients. Thus, identifying novel regulators of NST could provide new drug targets for anti-obesity and diabetes treatments. The mammalian target of rapamycin (mTOR) signaling network is a central regulator of cell growth and metabolism (Laplante & Sabatini, 2012; Dibble & Manning, 2013; Albert & Hall, 2014; Shimobayashi & Hall, 2014). mTOR is a highly conserved protein kinase found in two structurally and functionally distinct complexes named mTOR complex 1 (mTORC1) and mTORC2. mTORC1 is sensitive to the macrolide rapamycin and contains mTOR, mammalian lethal with sec-13 protein (mLST8), and regulatory associated protein of mTOR (raptor). mTORC2 is rapamycin insensitive and contains mTOR, mLST8, mammalian stress-activated map kinase-interacting protein 1 (mSIN1), and rapamycin-insensitive companion of mTOR (rictor). mTORC2 is activated by growth factors, such as insulin and insulin-like growth factor 1 (IGF-1), via phosphatidylinositol 3-kinase (PI3K)-dependent ribosome association (Zinzalla et al, 2011). mTORC2 downstream targets are members of the AGC kinase family, such as Akt, serum/glucocorticoid-regulated kinase (SGK), and protein kinase C (PKC) (Sarbassov et al, 2005; Jacinto et al, 2006; Garcia-Martinez & Alessi, 2008; Ikenoue et al, 2008; Cybulski & Hall, 2009), through which mTORC2 promotes lipogenesis, glucose uptake, glycolysis, and cell survival (Manning & Cantley, 2007; Kumar et al, 2008; Hagiwara et al, 2012; Yuan et al, 2012). Due to its role in mediating lipid and glucose homeostasis, dysfunction of mTORC2 signaling has been implicated in the development of insulin resistance and diabetes. Moreover, a recent study by Olsen et al (2014) demonstrated that mTORC2 in brown adipocytes in vitro mediates β-adrenergic stimulation-induced glucose uptake. However, a role for mTORC2 in thermogenesis, and in particular NST, has so far not been investigated. Here, we show that β-adrenergic stimulation and cold exposure activate mTORC2 signaling in brown adipocytes in vitro and in vivo. We find that mTORC2 in BAT stimulates cold-induced glucose uptake and glycolysis. Consequently, mice with adipose tissue-specific inactivation of mTORC2 (AdRiKO mice) are hypothermic and unable to maintain stable body temperature upon cold exposure. Restoration of either Akt signaling or glucose metabolism in BAT of AdRiKO mice restored body temperature and improved cold tolerance. Thus, mTORC2 in BAT is essential for maintenance of energy homeostasis and body temperature upon cold exposure. Results Norepinephrine activates mTORC2 in vitro via cAMP, PI3K, and Epac1 To investigate the role of mTORC2 signaling in NST, we first examined whether mTORC2 is activated by signals that induce thermogenesis. In particular, differentiated brown adipocytes (dBACs) were treated with NE to induce β-adrenergic signaling. As expected, NE treatment resulted in stimulation of PKA signaling as suggested by increased Creb-S133, HSL-S563, and perilipin phosphorylation (Fig 1A). Importantly, NE also stimulated phosphorylation of the mTORC2 target Akt at S473, a major readout of mTORC2 activity (Hresko & Mueckler, 2005; Sarbassov et al, 2005; Cybulski & Hall, 2009) (Fig 1A). Moreover, NE stimulation also induced phosphorylation of mTOR at S2481, another indicator of mTORC2 activation (Copp et al, 2009). These observations suggest that β-adrenergic stimulation induces mTORC2 signaling, in addition to PKA, in dBACs. Figure 1. NE activates mTORC2 in vitro via cAMP, PI3K, and Epac1 Immunoblot analysis of BAT cells stimulated with norepinephrine (NE) for the indicated proteins. Immunoblot analysis of BAT cells stimulated with NE for 5 min in the presence of rapamycin (Rapa), Torin, or wortmannin (Wrtm) for the indicated proteins. Immunoblot analysis of BAT cells stimulated with 8-Br-cAMP for 5 min in the presence of Rapa, Torin, or Wrtm for the indicated proteins. Immunoblot analysis of BAT cells stimulated with NE for 5 min in the presence of Wrtm, H89, or ESI-09 for the indicated proteins. Immunoblot analysis of BAT cells stimulated with 8-Br-cAMP for 5 min in the presence of Wrtm, H89, or ESI-09 for the indicated proteins. Data information: All experiments were performed in triplicates, and a representative replicate is presented. Download figure Download PowerPoint Next, we investigated the pathway via which NE stimulates mTORC2. Insulin activates mTORC2 in a PI3K-dependent but mTORC1-independent manner. We examined whether NE activates mTORC2 in a similar manner. We stimulated dBACs with NE in the presence of the pan-mTOR (mTORC1 and mTORC2) inhibitor Torin, the mTORC1-specific inhibitor rapamycin, or the PI3K inhibitor wortmannin. Similar to insulin-induced mTORC2 stimulation, NE-induced activation of mTORC2 was independent of mTORC1, since pretreatment of dBACs with rapamycin did not prevent induction of Akt-S473 phosphorylation upon NE stimulation (Fig 1B). In contrast, inhibition of mTOR with Torin or of PI3K with wortmannin prevented Akt-S473 phosphorylation (Fig 1B). Hence, NE-induced activation of mTORC2 in dBACs is dependent on PI3K and independent on mTORC1. NE stimulation leads to an increase in intracellular cAMP, which is crucial for NE-induced activation of PKA signaling (Cannon & Nedergaard, 2004). To test whether cAMP is required for NE-induced activation of mTORC2, we treated dBACs with the cell-permeable cAMP analogue 8-Br-cAMP. Similar to NE stimulation, 8-Br-cAMP treatment induced Akt-S473 phosphorylation in dBACs (Fig 1C). 8-Br-cAMP stimulated mTORC2 signaling when mTORC1 was blocked with rapamycin, but was no longer able to induce Akt-S473 phosphorylation when mTOR or PI3K was inhibited with Torin or wortmannin, respectively (Fig 1C). Thus, NE induces mTORC2 signaling via cAMP and PI3K. Importantly, in the presence of mTOR or PI3K inhibition, NE or 8-Br-cAMP still induced HSL-S563 phosphorylation (Fig 1B and C), indicating that inhibition of mTOR or PI3K does not affect PKA signaling. cAMP has several target proteins, two of which are PKA and Epac1. Epac1 mediates cAMP-induced activation of mTORC2 in prostate cancer cells (Misra & Pizzo, 2012) and thus might be involved in cAMP-induced stimulation of mTORC2 in BAT. To investigate whether PKA or Epac1 is required for NE- or cAMP-induced activation of mTORC2, we stimulated dBACs with NE or 8-Br-cAMP in the presence of the PKA inhibitor H89 or the Epac inhibitor ESI-09. Treatment of dBACs with H89 efficiently blocked NE- and 8-Br-cAMP-induced HSL-S563 phosphorylation, suggesting that PKA signaling is inhibited. ESI-09 treatment slightly reduced but did not block induction of HSL-S563 phosphorylation by NE or 8-Br-cAMP stimulation (Fig 1D and E). Interestingly, treatment of dBACs with H89 resulted in hyperphosphorylation of Akt-S473, suggesting that inhibition of PKA signaling does not impair activation of mTORC2 (Fig 1D and E). In contrast, treatment with the Epac inhibitor ESI-09 prevented NE- and 8-Br-cAMP-induced phosphorylation of Akt-S473, suggesting that NE activates mTORC2 via cAMP, Epac1, and PI3K (Fig 1D and E). Norepinephrine and cold activate mTORC2 in vivo We next assessed whether NE can stimulate mTORC2 signaling in BAT in vivo. To this end, we used AdRiKO mice (Cybulski et al, 2009) which are defective in mTORC2 signaling in both BAT and white adipose tissue (WAT) (Fig EV1A). AdRiKO mice display increased lean mass and elevated insulin-like growth factor 1 (IGF-1) levels upon high-fat diet (Cybulski et al, 2009). To avoid confounding effects due to this growth phenotype, we used young (10–14 weeks) AdRiKO mice fed a standard diet. Under such conditions, AdRiKO mice are not altered in body weight, body composition, and circulating IGF-1 levels (Fig EV1B–D). In line with our in vitro results, treatment of control mice with NE induced phosphorylation of the mTORC2 target Akt and of S2481 on mTOR (Fig 2A). Importantly, AdRiKO mice did not display induction of Akt-S473 phosphorylation in BAT upon NE stimulation (Figs 2B and EV1E). Hence, functional mTORC2 is required for Akt phosphorylation in BAT in response to NE. Since NE is released from sympathetic nerves upon cold exposure, we hypothesized that mTORC2 signaling in BAT could also be induced by cold stress. Similar to the results obtained with NE stimulation, cold exposure induced Akt-S473 and mTOR-S2481 phosphorylation in BAT of control mice (Figs 2C and EV1F). Again, this induction was dependent on functional mTORC2 signaling as Akt-pS473, mTOR-pS2481, and phosphorylation of the Akt target FoxO1 were not induced in BAT upon cold exposure of AdRiKO mice (Figs 2C and EV1F). In contrast to BAT, mTORC2 signaling was not induced in inguinal subcutaneous WAT (sWAT) upon cold exposure (Fig EV1G). Taken together, these data demonstrate that mTORC2 signaling is induced by NE and cold in BAT but not in sWAT. Click here to expand this figure. Figure EV1. AdRiKO mice do not display alterations in body weight, plasma IGF-1 and locomotor activity. Immunoblot analysis of BAT and sWAT of AdRiKO and control mice housed at 22°C for the indicated proteins. Body weight of AdRiKO and control mice housed at 22°C [n = 14 (control), n = 12 (AdRiKO)]. Body composition of AdRiKO and control mice housed at 22°C (n = 18/group). Plasma IGF-1 levels in AdRiKO and control mice housed at 22°C [n = 11 (control), n = 9 (AdRiKO)]. Quantification of Akt-pS473 band intensity relative to total Akt band intensity shown in Fig 2B (n = 3/group). Quantification of Akt-pS473 and mTOR-pS2481 band intensity relative to total Akt or total mTOR band intensity shown in Fig 2C (n = 6/group). Immunoblot analysis of sWAT of AdRiKO and control mice housed at 22 or 4°C for 2 h for the indicated proteins (n = 6/group, each lane represents a mix of 3 mice). Locomotor activity of AdRiKO and control mice housed at 22°C (n = 13/group). Body temperature loss of AdRiKO and control mice upon cold exposure with ad libitum access to food [n = 11 (control), n = 10 (AdRiKO)]. Cold-induced shivering of AdRiKO and control mice housed at 4°C for 4 h (n = 6/group). Data information: Data represent mean ± SEM. Statistically significant differences between AdRiKO and control mice were determined with unpaired Student's t-test and indicated with asterisks (*P < 0.05; **P < 0.01; ***P < 0.001). Statistically significant differences between temperatures or treatments were determined with unpaired Student's t-test and are indicated with a number sign (#P < 0.05; ##P < 0.01). The exact P-value for each significant difference can be found in Appendix Table S2. Download figure Download PowerPoint Figure 2. NE and cold activate mTORC2 in vivo Immunoblot analysis of BAT from control mice treated with either norepinephrine (NE) or vehicle for 30 min for the indicated proteins (n = 3/group). Immunoblot analysis of BAT from AdRiKO and control mice treated with either norepinephrine (NE) or vehicle for 30 min for the indicated proteins (n = 3/group). Immunoblot analysis of BAT from AdRiKO and control mice housed at either 22 or 4°C for 2 h for the indicated proteins (n = 6/group, each lane represents a mix of 3 mice). Body temperature of AdRiKO and control mice housed at 22°C [n = 11 (control), n = 9 (AdRiKO)]. Body temperature of AdRiKO and control mice housed at 30°C for 2 weeks (n = 8/group). Body temperature loss upon cold exposure of AdRiKO and control mice [n = 20 (control), n = 17 (AdRiKO)]. Data information: Data represent mean ± SEM. Statistically significant differences between AdRiKO and control mice were determined with unpaired Student's t-test and are indicated with asterisks (*P < 0.05; **P < 0.01; ***P < 0.001). The exact P-value for each significant difference can be found in Appendix Table S2. Download figure Download PowerPoint As we observed an induction of mTORC2 signaling in BAT upon NE and cold stimulation, we next investigated whether a defect in mTORC2 signaling affected temperature regulation. AdRiKO mice were hypothermic when housed at 22°C, which is a mild temperature stress for mice (Fig 2D). The hypothermia could not be accounted for by a reduction in locomotor activity (Fig EV1H). In contrast, housing AdRiKO mice at thermoneutrality (30°C) for 2 weeks prevented this hypothermic phenotype (Fig 2E). Next, we performed an acute cold exposure with AdRiKO and control mice. In contrast to control mice, AdRiKO mice were unable to maintain stable body temperature when housed at 4°C (Fig 2F). Interestingly, when food was provided during acute cold exposure, AdRiKO mice displayed a less severe loss of body temperature, but were still unable to maintain stable body temperature in the cold (Fig EV1I). Thus, inactivation of mTORC2 signaling in adipose tissue leads to decreased body temperature and increased sensitivity to cold stress, in particular under nutrient-limiting conditions. Cold-induced muscle shivering also contributes to heat generation upon acute cold exposure (Cannon & Nedergaard, 2004). To investigate whether the increased sensitivity to cold stress observed in AdRiKO mice was due to impaired shivering thermogenesis, we measured cold-induced muscle shivering in AdRiKO and control mice after 4-h cold exposure. Interestingly, cold-exposed AdRiKO mice showed significantly increased cold-induced muscle shivering compared to control mice (Fig EV1J). The increased shivering could be a compensatory reaction of the AdRiKO mice to maintain body temperature upon cold stress. mTORC2 in adipose tissue is not required for cold-induced lipid droplet mobilization, mitochondrial uncoupling, and β-oxidation Thermogenesis upon β-adrenergic stimulation requires mobilization of lipid stores, induction of β-oxidation, and stimulation of mitochondrial uncoupling to generate heat. Since AdRiKO mice are hypothermic and exhibit increased sensitivity to cold (see above), we investigated whether AdRiKO mice are defective in lipid mobilization, β-oxidation, or mitochondrial uncoupling in adipose tissue. There was no difference in BAT and sWAT weights between AdRiKO and control mice housed at either 22 or 4°C (Fig EV2A and B). Moreover, there was no discernible difference in the morphology of lipid droplets in sWAT from AdRiKO mice compared to wild-type control mice kept at 22°C. Furthermore, both control and AdRiKO mice were able to mobilize sWAT lipid stores upon cold exposure (4°C) as suggested by a reduction in the size of lipid droplets, that is, the appearance of multilocular adipocytes in sWAT (Fig 3A). In line with this, cold-exposed AdRiKO and control mice both displayed a significant increase in levels of circulating free fatty acids (NEFAs) and glycerol (Fig 3B and C). Even though the increase in circulating NEFAs was slightly less in AdRiKO mice compared to control mice, AdRiKO mice still displayed a twofold increase in circulating NEFAs upon cold exposure (Fig 3B). In BAT, AdRiKO mice housed at 22°C displayed larger lipid droplets compared to control mice (Fig 3D). However, at 4°C lipid droplet size in BAT decreased to the same extent in AdRiKO and control mice (Fig 3D). Despite the difference in lipid droplet size, we did not observe a significant difference in total triglyceride (TG) content in BAT upon cold exposure or between control and AdRiKO mice (Fig 3E). In contrast to this, free fatty acid levels in BAT were strongly enhanced in the AdRiKO mice upon cold exposure (Fig 3F). These findings suggest that the defect in temperature regulation in AdRiKO mice is most likely not due to decreased availability of free fatty acids upon cold exposure. Click here to expand this figure. Figure EV2. mTORC2 in adipose tissue does not affect BAT and sWAT weight. BAT weight of AdRiKO and control mice housed at 22 or 4°C for 8 h (n = 6/group). Data represent mean ± SEM. sWAT weight of AdRiKO and control mice housed at 22 or 4°C for 8 h (n = 6/group). Data represent mean ± SEM. Download figure Download PowerPoint Figure 3. mTORC2 in adipose tissue is not required for cold-induced lipid droplet mobilization Representative H&E staining of sWAT sections from AdRiKO and control mice (n = 5/group). Non-esterified fatty acids (NEFAs) in plasma of AdRiKO and control mice (n = 6/group). Glycerol in plasma of AdRiKO and control mice (n = 6/group). Representative H&E staining of BAT sections from AdRiKO and control mice (n = 5/group). Triglycerides (TGs) in BAT of AdRiKO and control mice housed at 22 or 4°C for 8 h (n = 6/group). NEFAs in BAT of AdRiKO and control mice (n = 6/group). Data information: Data represent mean ± SEM. Statistically significant differences between AdRiKO and control mice were determined with unpaired Student's t-test and are indicated with asterisks (**P < 0.01; ***P < 0.001). Statistically significant differences between temperatures were determined with unpaired Student's t-test and are indicated with a number sign (#P < 0.05; ##P < 0.01; ###P < 0.001). The exact P-value for each significant difference can be found in Appendix Table S2. Download figure Download PowerPoint Since cold-exposed AdRiKO mice display significantly increased levels of free fatty acids in BAT compared to control mice (Fig 3F), we hypothesized that this increase in NEFAs might be due to impaired mitochondrial function, which could lead to accumulation of NEFAs in BAT. To test this possibility, we first measured induction of genes involved in mitochondrial uncoupling in BAT upon cold exposure. Despite the cold-sensitive phenotype of AdRiKO mice, mRNA levels of UCP1, Dio2, and PGC-1α were induced to a similar extent in BAT of AdRiKO and control mice (Fig 4A), and UCP1 protein levels in BAT were also similar (Fig 4B). Second, AdRiKO mice did not exhibit any defect in expression of genes involved in β-oxidation (Fig 4C). Thus, AdRiKO mice appear normal for induction of the thermogenic transcriptional program and expression of β-oxidation genes. Third, we measured expression of proteins of the electron transport chain in BAT. AdRiKO mice displayed a slight decrease (22°C) or no change (4°C) in expression of electron transport chain proteins compared to control mice (Fig 4D). Fourth, mitochondrial DNA (mtDNA) copy number was unchanged in BAT of AdRiKO mice (Fig 4E), suggesting that BAT of AdRiKO and control mice contain a similar amount of mitochondria. Fifth, EM micrographs of BAT revealed no difference between AdRiKO and control mitochondria with regard to size, shape, and cristae structure (Fig 4F). Finally, cold-exposed AdRiKO mice exhibited normal induction of oxygen consumption in BAT (Fig 4G) and at the whole-body level (Fig 4H). Thus, BAT in AdRiKO mice has normal mitochondrial function and oxidative metabolism can be efficiently induced upon cold stress. This suggests that the observed cold sensitivity of AdRiKO mice does not stem from a mitochondrial defect. Figure 4. mTORC2 in adipose tissue is not required for cold-induced mitochondrial uncoupling and β-oxidation mRNA levels of the indicated genes in BAT of AdRiKO and control mice housed at 22 or at 4°C for 8 h (n = 6/group). Immunoblot analysis of BAT from AdRiKO and control mice housed at 22 or at 4°C for 8 h for the indicated proteins (n = 6/group, each lane represents a mix of 3 mice). mRNA levels of the indicated genes in BAT of AdRiKO and control mice housed at 22 or at 4°C for 8 h (n = 6). Immunoblot analysis of BAT from AdRiKO and control mice housed at 22 or at 4°C for 8 h for the indicated proteins (n = 6/group, each lane represents a mix of 3 mice). Mitochondrial DNA content of BAT from AdRiKO and control mice housed at 22 or at 4°C for 8 h (n = 6/group). Representative electron micrographs of BAT from AdRiKO and control mice housed at 22 or at 4°C for 4 h (n = 3/group). Oxygen consumption rate (OCR) of BAT explants from AdRiKO and control mice housed at 22 or at 4°C for 4 h (n = 7/group). Maximal respiration (VO2 max) of AdRiKO and control mice housed at 22 or at 4°C for 8 h [n = 9 (control 22°C), n = 7 (AdRiKO 22°C), n = 8 (control 4°C), n = 8 (AdRiKO 4°C)]. Respiration (VO2) of AdRiKO and control mice upon cold exposure (n = 8/group). Data information: Data represent mean ± SEM. Statistically significant differences between AdRiKO and control mice were determined with unpaired Student's t-test and are indicated with asterisks (*P < 0.05; ***P < 0.001). Statistically significant differences between temperatures were determined with unpaired Student's t-test and are indicated with a number sign (#P < 0.05; ##P < 0.01; ###P < 0.001). The exact P-value for each significant difference can be found in Appendix Table S2. Download figure Download PowerPoint Despite a similar maximal induction of whole-body respiration (Fig 4H), AdRiKO mice were unable to maintain an enhanced metabolic rate throughout the duration of the cold exposure time course (Fig 4I). This inability to maintain an enhanced metabolic rate may account for the inability of AdRiKO mice to sustain an NST response. mTORC2 in adipose tissue is required for cold-induced glucose uptake and glycolysis Glucose uptake and glycolysis are strongly enhanced in BAT upon cold exposure, to compensate for the loss of mitochondrial ATP production due to heat-generating mitochondrial uncoupling (Greco-Perotto et al, 1987; Vallerand et al, 1990; Hao et al, 2015). Moreover, glucose is also needed for anaplerotic reactions to maintain fatty acid oxidation, and to generate glycerol 3-phosphate for lipid synthesis. mTORC2 is an important regulator of insulin-induced glucose uptake and glycolysis in WAT, muscle, and liver (Kumar et al, 2