Better explicating the strengths and shortcomings of these models will help refine projections and improve transparency in the years ahead.
Image credit: Witsawat.S.
Edited by Michael N. Hall, University of Basel, Basel, Switzerland, and approved November 3, 2014 (received for review June 25, 2014)
Significance
Adipose tissue maintains metabolic homeostasis during fasting and fed conditions. When nutrients are plentiful, anabolic signaling is mediated by insulin, stimulating adipocytes to take up glucose for energy storage. In the absence of nutrients, catabolic signaling initiates lipolysis, or the release of lipids for energy use, and is mediated by catecholamines. These opposing pathways are evolutionarily conserved and prevent futile cycling, but can lead to metabolic disorders such as insulin resistance if not properly regulated. Here we define a novel mechanism whereby lipolysis inhibits insulin-stimulated glucose uptake in adipocytes. This signaling mechanism likely contributes to insulin resistance when lipolysis is active, such as during high stress or obesity, and this new understanding may lead to novel treatment approaches for hyperglycemia.
Abstract
Anabolic and catabolic signaling oppose one another in adipose tissue to maintain cellular and organismal homeostasis, but these pathways are often dysregulated in metabolic disorders. Although it has long been established that stimulation of the β-adrenergic receptor inhibits insulin-stimulated glucose uptake in adipocytes, the mechanism has remained unclear. Here we report that β-adrenergic–mediated inhibition of glucose uptake requires lipolysis. We also show that lipolysis suppresses glucose uptake by inhibiting the mammalian target of rapamycin (mTOR) complexes 1 and 2 through complex dissociation. In addition, we show that products of lipolysis inhibit mTOR through complex dissociation in vitro. These findings reveal a previously unrecognized intracellular signaling mechanism whereby lipolysis blocks the phosphoinositide 3-kinase–Akt–mTOR pathway, resulting in decreased glucose uptake. This previously unidentified mechanism of mTOR regulation likely contributes to the development of insulin resistance.
Adipose tissue plays an essential role in maintaining whole-body energy homeostasis by storing or releasing nutrients. This balance is controlled by opposing signaling pathways where anabolic processes are activated by insulin (INS) and catabolic actions are activated by catecholamines. An important unanswered question in adipose biology is how catecholamine-induced β-adrenergic signaling opposes insulin-stimulated glucose uptake (1⇓⇓⇓⇓–6). Surprisingly, the underlying mechanism for this well-established physiological response in adipocytes is still unknown.
When nutrients are plentiful, insulin is released by the pancreas and stimulates the absorption of glucose and fatty acids in adipose tissue, where they are packaged and stored as triacylglycerol (TAG) in cellular lipid droplets. Insulin signaling in adipocytes is mediated by the phosphoinositide 3-kinase (PI3K)–Akt–mTOR pathway. mTOR is a highly conserved serine/threonine protein kinase that functions in either of two distinct multiprotein complexes, mTOR complex 1 (mTORC1) and mTOR complex 2 (mTORC2). mTORC1 is defined primarily by the association of mTOR with raptor, whereas mTORC2 includes mTOR with rictor (7). Importantly, mTORC2 phosphorylation of Akt at S473 is required for Akt activity on AS160, which is necessary for glucose uptake in response to insulin (8⇓⇓–11). Of note, for both mTORC1 and mTORC2, the integrity of these protein complexes is essential for kinase substrate specificity and proper signaling (12, 13).
During periods of fasting or stress, catecholamines are released by the sympathetic nervous system to activate lipolysis. Stimulation of the β-adrenergic receptor on adipocytes activates adenylyl cyclase (AC), leading to elevated cAMP and protein kinase A (PKA) activity. PKA initiates lipolysis by direct phosphorylation of hormone-sensitive lipase (HSL) and perilipin (14⇓–16) and indirect activation of adipose triglyceride lipase (ATGL) (17⇓–19). Lipolysis involves hydrolysis of TAG stored in the lipid droplet to produce diacylglycerol (DAG), monoacylglycerol (MAG), fatty acids, and glycerol. These lipolytic products are important energy substrates that can act as precursors for other lipids and impact cellular signaling. However, their potential role as signaling molecules has been underappreciated (20).
In this study, we provide insight into the mechanisms that link β-adrenergic stimulation to the inhibition of insulin-stimulated glucose uptake. Namely, we show that activation of lipolysis is crucial. Moreover, we find that products of lipolysis themselves cause mTOR inhibition by complex dissociation, which inhibits glucose uptake in adipocytes. This mechanism of mTOR regulation (i.e., by complex dissociation) has major implications in the regulation of cellular metabolism and likely contributes to stress-induced hyperglycemia and obesity-induced insulin resistance.
Results
Catecholamine-Induced Inhibition of Glucose Uptake and Insulin Signaling Requires Lipolysis.
Mice lacking ATGL show improved glucose tolerance and are resistant to high-fat diet–induced insulin resistance (21⇓–23). ATGL deficiency also improves insulin signaling in white adipose tissue (21). These observations implicate lipolysis in insulin resistance. In addition, stimulation of the β-adrenergic receptor in isolated adipocytes is known to acutely inhibit insulin-stimulated glucose uptake (2, 3, 6). To investigate a possible role for lipolysis in the effects of catecholamine action, we compared glucose uptake in WT and ATGL−/− primary mouse adipocytes during treatment with isoproterenol, a β-adrenergic receptor agonist. As previously reported, isoproterenol inhibited glucose uptake in WT adipocytes; however, here we show that this inhibition was rescued in the absence of ATGL (Fig. 1A and Fig. S1A). As expected, lipolysis was effectively blocked in ATGL−/− adipocytes even during treatment with isoproterenol (Fig. S1B). Rescue of glucose uptake was also observed in cultured 3T3-L1 adipocytes upon lipase inhibition with E600, a general lipase inhibitor that binds irreversibly to the active site of lipases (Fig. S1C). In addition, insulin signaling necessary for GLUT4 translocation was restored in the ATGL−/− adipocytes compared with WT (Fig. 1B and Fig. S1 D and E). Taken together, these data show that lipolysis is required for catecholamine-mediated inhibition of glucose uptake in adipocytes.
Fig. 1.
Catecholamine-induced inhibition of glucose uptake and insulin signaling requires lipolysis. (A) Radiolabeled glucose uptake assay in WT vs. ATGL−/− primary mouse adipocytes. Isolated adipocytes were treated with or without insulin (10 nM) in the presence or absence of isoproterenol (0.1 μM) for 30 min followed by [U-14C]-d-glucose (10 μM) for 20 min. All assays contained adenosine deaminase (ADA) (2 units/mL). (B) Western blot analysis of insulin signaling in WT and ATGL−/−primary mouse adipocytes treated as in A before the addition of glucose. Each graph represents mean ± SEM from triplicate experiments. Asterisks indicate significant difference (***P < 0.001).
The β-Adrenergic/cAMP Pathway Impairs Insulin Signaling by Inhibiting the mTOR Complexes.
Similar to isoproterenol-mediated inhibition of insulin signaling, treating adipocytes with forskolin, a potent activator of AC, inhibits insulin signaling (4, 5). We found that forskolin action significantly inhibited mTORC1 and -2 in response to insulin as measured by phosphorylation of S6K (T389) and Akt (S473), respectively (Fig. 2A). Although signaling downstream of the mTOR complexes was inhibited, upstream signaling was unaffected, as shown by tyrosine phosphorylation of the insulin receptor and of PDK1-mediated phosphorylation of Akt at T308 (Fig. 2A). Analogous results were observed in primary rat adipocytes treated with isoproterenol before insulin stimulation (Fig. S2A), and the inhibition of AS160 in both cultured 3T3-L1 and primary rat adipocytes (Fig. 2A and Fig. S2A) demonstrates that the AC activity plays a role in dampening glucose uptake. The effects of AC activity on mTOR were reiterated by treatment with a membrane permeable cAMP analog cpt-cAMP, the phosphodiesterase (PDE) inhibitor 3-isobutyl-1-methylxanthine (IBMX), and isoproterenol, demonstrating that elevated cAMP is sufficient to inhibit mTOR in adipocytes (Fig. 2B and Fig. S2B). Activation of adipocyte lipolysis was measured by phosphorylation of HSL (Fig. 2 A and B and Fig. S2A) and glycerol release (Fig. S2 C and D). In addition, treatment with forskolin had no effect on phosphorylation of raptor by AMPK (Fig. S2E). Interestingly, forskolin does not inhibit insulin signaling in 3T3-L1 fibroblasts before differentiation into adipocytes or in primary mouse hepatocytes (Fig. S2F). Taken together, these data demonstrate that cAMP activity inhibits mTOR in adipocytes, which may play a role in the observed catecholamine-induced decrease in glucose uptake.
Fig. 2.
The β-adrenergic/cAMP pathway impairs insulin signaling by inhibiting the mTOR complexes. (A) Western blot and quantitative analysis of cultured 3T3-L1 adipocytes treated with or without forskolin (FSK, 10 μM), rapamycin (Rap, 20 nM), or Torin1 (250 nM) for 30 min before insulin treatment (INS, 10 nM) for 15 min. (B) Western blot and quantitative analysis of cultured 3T3-L1 adipocytes treated with or without forskolin (FSK, 10 μM), Cpt-cAMP (Cpt, 100 μM), isoproterenol (ISO, 5 μM), or IBMX (200 μM) for 30 min before insulin treatment (INS, 10 nM) for 15 min. Each graph represents mean ± SEM from triplicate experiments. Asterisks indicate significant difference (*P < 0.05 or **P < 0.01, respectively).
cAMP-Mediated Inhibition of mTOR Requires PKA and Lipase Activity.
The primary role of cAMP in adipocytes is to activate PKA, which leads to lipolysis through lipase activation and phosphorylation of perilipin (14⇓–16). To investigate the mechanism of cAMP-mediated inhibition of mTOR, we pharmacologically inhibited PKA during forskolin and insulin treatment. The ATP-competitive PKA inhibitor, H89, greatly inhibited PKA activity and rescued both mTORC1 and -2 activity in response to insulin (Fig. 3 A and C). In addition, blocking lipase action with E600; atglistatin, a specific ATGL inhibitor; or CAY10499, a specific HSL inhibitor, reversed the cAMP-mediated inhibition of mTOR (Fig. 3 B and C and Fig. S3A). Interestingly, blocking lipase action induced S6K phosphorylation during treatment with forskolin alone (Fig. 3B), whereas adipocytes have consistently shown mTORC1 inhibition in response to elevated cAMP (24⇓–26). This seemingly paradoxical trend may be similar to cAMP activation of mTORC1 in other cell types (27), when the inhibitory effects of lipolysis are absent. This was not observed in mTORC2 activity on Akt (Fig. 3B). Lipase inhibition also blocked lipolysis as measured by glycerol release (Fig. S3B). Taken together, these data demonstrate that elevated cAMP acts through PKA and activation of lipolysis to disrupt mTOR activity in adipocytes.
Fig. 3.
cAMP-mediated inhibition of mTOR requires PKA and lipase activity. (A) Western blot analysis of cultured 3T3-L1 adipocytes pretreated with H89 (10 μM) for 20 min before forskolin and insulin treatment as in Fig. 2A. (B) Western blot analysis from cultured 3T3-L1 adipocytes pretreated with diethyl-p-nitrophenylphosphate (E600, 150 μM) or atglistatin (10 μM) before forskolin and insulin treatment as in Fig. 2A. (C) Quantitative analysis of pS6K (T389) and pAKT (S473) from experiments in A and B. Each graph represents mean ± SEM from triplicate experiments. Asterisks indicate significant difference (*P < 0.05 or **P < 0.01, respectively).
Lipolytic Products Inhibit mTOR Activity in Vitro.
Lipolysis produces DAG, MAG, fatty acid, and glycerol through lipase action on TAG. To determine if these lipolytic products are responsible for lipolysis-mediated inhibition of mTOR, we examined their ability to inhibit purified recombinant mTOR (Fig. 4A) in vitro. We extracted lipids from adipocytes treated with or without the lipolytic agents forskolin or isoproterenol and included these lipids in mTOR kinase assays. We show that lipids extracted from cultured adipocytes undergoing lipolysis consistently inhibited mTOR, whereas lipids from control cells had no effect (Fig. 4B and Fig. S4A). Lipids extracted from WT primary mouse adipocytes showed similar results; however, lipids from either control or isoproterenol-treated ATGL−/− mouse adipocytes had no effect on mTOR (Fig. 4C). Importantly, although the lipids extracted from control cells did not inhibit mTOR, treating the lipids with a lipase in vitro did generate inhibitory lipids (Fig. 4D). To investigate what lipids may be responsible for inhibiting mTOR, we included specific fatty acids or glycerolipids in the mTOR kinase assay. These showed no significant effect on mTOR activity (Fig. 4E), suggesting that it may be a particular lipid released during lipolysis and not lipolytic products in general that inhibit mTOR. To verify the kinase activity was solely due to purified mTOR, we show complete inhibition by Torin1 (Fig. S4B) and kinetic analysis of mTORC1 activity in vitro (Fig. S4C). These data demonstrate that lipolytic products inhibit mTOR in vitro, suggesting that they may facilitate catecholamine-induced inhibition of glucose uptake in adipocytes.
Fig. 4.
Lipolytic products inhibit mTOR activity in vitro. (A) Coomassie stain of recombinant mTORC1 purification showing mTOR (upper band) and raptor (lower band). (B) Radioactive in vitro mTOR kinase assays using purified recombinant mTORC1 and 4E-BP1 as substrate. No lipid vehicle or lipids extracted from cultured 3T3-L1 adipocytes treated with or without forskolin (FSK, 10 μM) for 30 min were added to the assay 10 min before the addition of [γ-32P]-ATP. (C) mTOR kinase assays as in B using lipids extracted from either WT or ATGL−/− primary mouse adipocytes after treatment with or without isoproterenol (ISO, 10 μM) for 30 min. (D) mTOR kinase assays as in B using lipids extracted from cultured 3T3-L1 adipocytes. The lipids were treated with or without lipase in vitro before adding them to the mTOR kinase assay. (E) mTOR kinase assays as in B where DAG (specifically, 1-palmitoyl-2-oleoyl-sn-glycerol), MAG (specifically, 2-oleoyl-glycerol), oleate, or palmitate were added as indicated. Each graph represents mean ± SEM from triplicate experiments. Asterisks indicate significant difference (*P < 0.05 or **P < 0.01, respectively).
Lipolytic Products Inhibit mTOR Through Complex Dissociation.
In our attempts to purify mTOR from adipocytes, we observed that the mTOR complexes 1 and 2 were dissociated in cells treated with forskolin compared with control (Fig. 5A and Fig. S5A). Similar to the rescue of mTOR signaling shown in Fig. 3 B and C, atglistatin rescued mTORC1 and -2 association in cultured adipocytes (Fig. 5B), suggesting that the mechanism of forskolin-induced mTOR inhibition occurs through mTOR complex dissociation. Complementary to the glucose uptake and insulin signaling data in Fig. 1, mTORC2 dissociation was also observed in WT and rescued in ATGL−/− primary mouse adipocytes (Fig. 5C). These data demonstrate that lipolysis is required for the observed mTOR complex dissociation. In addition, lipid extracts from cultured adipocytes treated with forskolin caused mTOR complex dissociation in vitro, whereas lipids from vehicle-treated adipocytes had no effect on the complex (Fig. S5B). To quantitatively show mTOR dissociation in vitro, we purified a fluorescently tagged mTOR complex composed of a Venus-tagged mTOR and a Cerulean-tagged raptor or rictor (Fig. 5D). This recombinant mTOR complex was useful because fluorescent protein tags can be spectrophotometrically detected. In our mTOR dissociation assay, the detected Venus or Cerulean emission directly represents the presence of mTOR or raptor/rictor, respectively (Fig. S5C), and dissociation can be efficiently and quantitatively determined in vitro. We used this assay to show that lipids extracted from adipocytes treated with forskolin dissociate mTORC1 and -2 in vitro, whereas lipids extracted from untreated cells, metabolites that partition to the aqueous phase, or lipids from 3T3-L1 fibroblasts before differentiation or primary hepatocytes have no effect (Fig. 5E). Complementary to the insulin signaling shown in Fig. 3, lipase inhibition also blocked the dissociation of mTOR (Fig. 5F). Lipids produced from in vitro lipase treatment of adipocyte cell extracts also caused mTOR dissociation, whereas vehicle-treated lipids did not (Fig. 5G). Taken together, these data show that lipolytic products facilitate mTOR inhibition through mTOR complex dissociation.
Fig. 5.
Lipolytic products inhibit mTOR through complex dissociation. (A) Western blot analysis of mTORC1 and mTORC2 coimmunoprecipitations against raptor and rictor, respectively, where cultured adipocytes were treated with or without forskolin (FSK, 10 μM), rapamycin (Rap, 20 nM), or Torin1 (250 nM) for 30 min before insulin treatment (INS, 10 nM) for 15 min. (B) Western blot analysis of mTORC1 and mTORC2 coimmunoprecipitations against mTOR, where cultured adipocytes were treated with or without atglistatin (10 μM) for 1 h before treatment with forskolin and insulin as in A. (C) Western blot analysis of mTORC2 association using coimmunoprecipitations against mTOR, where WT or ATGL−/− primary mouse adipocytes were treated with or without insulin (10 nM) in the presence or absence of isoproterenol (0.1 μM) for 30 min as in Fig. 1 A and B before addition of glucose. (D) Illustrative representation of the mTOR dissociation assay. (E) mTOR dissociation assay where cultured 3T3-L1 adipocytes were treated with or without forskolin (10 μM) before cell lysis and organic extraction (Materials and Methods). Purified fluorescently tagged mTORC1 or mTORC2 was incubated with either lysate or extracted organic or aqueous phases from cultured adipocytes or lysate from fibroblasts or hepatocytes, for 30 min before washing and detection of fluorescence. (F) mTOR dissociation assay where cultured 3T3-L1 adipocytes were treated with DMSO as control, diethyl-p-nitrophenylphosphate (E600, 150 μM), or atglistatin (10 μM) before forskolin treatment and incubation with fluorescent mTOR as in E. (G) mTOR dissociation assay where lipids were extracted from cultured 3T3-L1 adipocytes and treated with or without lipase in vitro, then incubated with mTOR as in E. Each graph represents mean ± SEM from triplicate experiments. # indicates significant difference from control (#P < 0.0001).
Torin1-Induced Inhibition of Glucose Uptake Is Independent of Lipolysis.
mTOR activity is necessary to facilitate insulin-induced glucose uptake in adipocytes (8⇓⇓–11). Here we show that isoproterenol stimulation of the β-adrenergic receptor and direct mTOR inhibition by Torin1 inhibit glucose uptake in WT primary mouse adipocytes, whereas direct mTOR inhibition alone is sufficient to block glucose uptake in the absence of ATGL (Fig. 6A). In addition, whereas stimulation of the β-adrenergic receptor only inhibits insulin signaling in WT adipocytes (Fig. 1B and Fig. S1 D and E), Torin1 inhibits insulin signaling in both WT and ATGL−/− adipocytes (Fig. 6B). Taken together, these data suggest that mTOR inhibition by lipolysis is a likely mechanism of catecholamine-induced inhibition of glucose uptake in adipocytes.
Fig. 6.
Torin1-induced inhibition of glucose uptake is independent of lipolysis. (A) Radiolabeled glucose uptake assay in WT vs. ATGL−/− primary mouse adipocytes. Isolated adipocytes were treated with or without Torin1 (250 nM) for 10 min before treatment with or without insulin (10 nM) in the presence or absence of isoproterenol (0.1 μM) for 30 min, followed by the addition of [U-14C]-d-glucose (10 μM) for 20 min. All assays contained adenosine deaminase (ADA) (2 units/mL). (B) Western blot analysis of insulin signaling in WT and ATGL−/−primary mouse adipocytes treated as in A before the addition of glucose. (C) Illustration of the proposed mechanism linking β-adrenergic receptor stimulation to impaired glucose uptake in adipocytes. Each graph represents mean ± SEM from triplicate experiments. Asterisks indicate significant difference (***P < 0.001).
Discussion
The major finding of this study is that lipolysis acutely inhibits insulin-stimulated glucose uptake in adipocytes. The ability of catecholamines to dampen adipocyte glucose uptake has been known for decades and documented repeatedly (2⇓⇓⇓–6), but the mechanism has remained unclear. Here we not only show that lipolysis is required to mediate this inhibition of glucose uptake, but that the mechanism of inhibition may be through dissociation of the mTOR complexes and subsequent inhibition of insulin-stimulated Akt activity. Interestingly, mTOR signaling and complex association are rescued when lipolysis is inhibited, and lipolytic products that partition to the organic phase are able to directly dissociate mTOR in vitro, suggesting that these lipids can act as signaling molecules to regulate insulin action. Lipolytic products produced in vitro also inhibit mTOR through dissociation, suggesting that further enzymatic activity is not required for lipolysis to mediate these effects on mTOR. Although it has recently been shown that lipolytic products can activate peroxisome proliferator-activated receptor (PPAR) α and δ (28), potentially impacting insulin signaling through PTEN expression, our finding that insulin signaling upstream of mTOR is unaffected by lipolysis suggests this is not the mechanism of insulin resistance in our model.
Lipotoxicity is one of the hypotheses being explored to explain the mechanisms by which obesity induces insulin resistance. Also known as the lipid metabolite theory, lipotoxicity is characterized by an excess of lipids that can act as signaling molecules to inhibit insulin signaling (29⇓–31). Previous data have shown that insulin resistance is often associated with lipid accumulation in liver and skeletal muscle, and high levels of circulating lipolytic products may correlate with obesity and insulin resistance in type 2 diabetes (32, 33). Importantly, lipid accumulation in the liver has been shown to inhibit insulin signaling specifically by decreasing mTORC2 complex integrity and activity (34). In addition, levels of basal lipolysis are elevated during obesity (35, 36) and decreased lipolysis due to lipase deficiency in mice attenuates diet-induced insulin resistance (22, 37). Taken together, these observations implicate lipolytic products in the development of insulin resistance, and here we demonstrate that lipids released during lipolysis have local effects on mTOR signaling and insulin-stimulated glucose uptake in adipocytes.
Although insulin resistance depends on insulin action in multiple tissues, adipose-specific GLUT4−/− mice develop systemic insulin resistance and hyperglycemia (38), whereas adipose-specific GLUT4 overexpression results in enhanced insulin sensitivity in vivo (39). This indicates that impaired insulin action in adipose tissue alone is sufficient to drive whole-body insulin resistance and hyperglycemia. Our previous work also demonstrates that impaired insulin action due to decreased rictor expression in adipose tissue results in whole-body insulin resistance and hyperglycemia (11). Taken together, our work shows mTOR complex inhibition as an acutely regulated event that leads to impaired insulin signaling in adipocytes, which may have an impact on the development of systemic insulin resistance. This study also demonstrates a previously unidentified mechanism of mTOR complex dissociation and inhibition by lipolytic products. This mechanism highlights the importance of lipolysis in regulating cellular signaling events, as a potential consequence of releasing energy stores.
In addition to mechanistic details of the opposing actions of anabolic and catabolic signaling in adipose tissue, our findings directly implicate lipolysis as the mechanism underlying adipose insulin resistance during acute stress events and may provide insight into obesity-induced insulin resistance. Acute hyperglycemia often develops after trauma or major surgery, particularly surgery within the abdominal cavity (40), after severe burn injuries, or sepsis. If untreated, this condition, termed stress-induced hyperglycemia, contributes to mortality and delays healing in postsurgery and patients in intensive care units (41). Although the effects of stress on insulin action are well known, the mechanistic link has remained unclear. The stress response is an evolutionary adaptation that conserves glucose for vital tissues, such as the brain, during injury. This response is characterized by rapid activation of the neuroendocrine and inflammatory systems to cause a metabolic state of stress. As a result, anabolic processes are inhibited, whereas catabolic processes, such as lipolysis, are enhanced to release substrates for tissue healing (41⇓–43). Our finding that lipolysis plays a key role in decreasing insulin signaling suggests that it may be a contributing factor in the development of stress-induced hyperglycemia.
In obese humans, whereas catecholamine stimulation of lipolysis is sometimes dampened, basal levels of lipolysis are commonly elevated. Therefore, in addition to the acute events investigated within this report, it is intriguing to consider the role of lipolysis and inhibition of the mTOR signaling pathways in adipose tissue in the development of obesity-induced insulin resistance over time. In addition to β-adrenergic stimulation, lipolysis is elevated by inflammatory cytokines, natriuretic peptides, growth hormones, and cortisol (44), highlighting that many factors could be contributing to this mechanism of insulin resistance. Although we have shown that acute stimulation of lipolysis may inhibit glucose uptake through mTOR complex dissociation, further investigation is necessary to determine the contribution of lipolysis to obesity-induced insulin resistance.
In this study, we have identified a novel mechanism of adipocyte signaling whereby lipolytic products dissociate the mTOR complexes, resulting in decreased insulin-stimulated glucose uptake (Fig. 6C). This model has implications in obesity-induced insulin resistance and stress-induced hyperglycemia and demonstrates that lipolytic products can function as signaling molecules to regulate cellular processes. It also provides insight into the mechanisms of opposing regulation between anabolic and catabolic signaling in adipose tissue.
Materials and Methods
Lipid Extraction and mTOR Dissociation in Vitro.
The 3T3-L1 adipocytes from 6-cm plates or 100 µL of packed isolated adipocytes were washed twice with PBS, homogenized in 100 µL buffer A (1 mM EDTA, 1 mM EGTA, 1 mM DTT, 0.1% Tween 20, 10 mM sodium phosphate, and 50 mM β-glycerophosphate, pH 7.4), supplemented with 1 mM phenylmethylsulfonyl fluoride, 10 μg/mL leupeptin, 10 μg/mL aprotinin, 10 μg/mL pepstatin, and 0.5 μm microcystin LR and homogenates were centrifuged at 16,000 × g for 10 min. A total of 400 μL of hexane/ethyl acetate (1:1) was added and mixed with the supernatants for 30 min followed by centrifugation at 8,000 × g for 2 min. The aqueous and organic phases were separated and the organic solvent was evaporated. The dried lipid residue was solubilized by suspension in buffer A (containing 0.1% Tween 20) and mixed with fluorescently tagged mTOR–Raptor or mTOR–Rictor immune complex on beads for 30 min at room temperature. The beads were then washed three times, transferred to a 96-well plate, and Venus and Cerulean emissions were detected. For the in vitro lipase treatment, resuspended lipids were treated with lipase (2 units/mL) for 30 min at room temperature and lipids were reextracted as described. The recombinant fluorescently tagged mTOR–Raptor and mTOR–Rictor complexes were generated by transient transfection into HEK293T cells using Lipofectamine 2000 (Invitrogen). HA–Venus–mTOR and FLAG–Cerulean–Raptor/FLAG–Cerulean–Rictor plasmids were transfected using 40 and 10 μg of plasmid/15-cm plate, respectively, and Lipofectamine 2000 at a 2:1 ratio of DNA:Lipofectamine.
Statistical Analysis.
Values are expressed as means ± SEM. Comparisons between two groups of the same treatment (insulin stimulated) were determined using Student t test. Comparisons among more than two groups were determined by one-way ANOVA with Dunnett’s post hoc analysis (INS set as the control). Data are represented in triplicate from three separate experiments unless otherwise indicated. Significance is indicated by *P < 0.05, **P < 0.01, ***P < 0.001, or #P < 0.0001, respectively.
Antibody reagents, cell culture, Western blot and quantitative analysis, animal care, primary adipocyte isolation, glucose uptake, mTOR immunoprecipitation, mTOR purification, mTOR kinase assay, and glycerol, glycerolipid, and fatty acid measurements are detailed in SI Materials and Methods.
Acknowledgments
We thank Evan Taddeo for isolating primary mouse hepatocytes. This research was funded by an American Diabetes Association Junior Faculty Award 7-11-JF-21 and R01 1R01DK101946 (to T.E.H.), R01 R01DK096076 (to N.L.), grants from the Belgian Fonds de la Recherche Scientifique (to P.P.R.), American Heart Association Predoctoral Fellowship 14PRE20480252 (to G.R.M.), and Télévie fellowships (to S. Blancquaert).
Footnotes
- ↵1To whom correspondence may be addressed. Email: teh3c{at}virginia.edu or lw6j{at}virginia.edu.
Author contributions: G.R.M., L.W., and T.E.H. designed research; G.R.M., L.W., V.R., S.G.S., R.C.G., S. Boroda, J.M.E., S. Blancquaert, and T.E.H. performed research; G.R.M., L.W., and T.E.H. contributed new reagents/analytic tools; G.R.M., L.W., P.P.R., N.L., and T.E.H. analyzed data; and G.R.M. and T.E.H. wrote the paper.
The authors declare no conflict of interest.
This article is a PNAS Direct Submission.
This article contains supporting information online at www.pnas.org/lookup/suppl/doi:10.1073/pnas.1410530111/-/DCSupplemental.
References
Lipolysis inhibits adipocyte glucose uptake
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