Egr1 mediates the effect of insulin on leptin transcription in adipocytes.

In mammals, leptin production in adipocytes is up-regulated by feeding and insulin. Although this regulatory connection is central to all physiological effects of leptin, its molecular mechanism remains unknown. Here, we show that the transcription factor early growth response 1, Egr1, is rapidly but transiently induced by insulin in adipose cells both in vitro and in vivo, and its induction is followed by an increase in leptin transcription. ChIP and luciferase assays demonstrate that Egr1 directly binds to and activates the leptin promoter. Interestingly, the lipid droplet protein FSP27 may work as a co-factor for Egr1 in regulating leptin expression. By using siRNA-mediated knockout of Egr1 along with its overexpression in adipocytes, we demonstrate that Egr1 is both necessary and sufficient for the stimulatory effect of insulin on leptin transcription.

4E-BPs Control Fat Storage by Regulating the Expression of Egr1 and ATGL.

Early growth response transcription factor Egr1 controls multiple aspects of cell physiology and metabolism. In particular, Egr1 suppresses lipolysis and promotes fat accumulation in adipocytes by inhibiting the expression of adipose triglyceride lipase. According to current dogma, regulation of the Egr1 expression takes place primarily at the level of transcription. Correspondingly, treatment of cultured adipocytes with insulin stimulates expression of Egr1 mRNA and protein. Unexpectedly, the MEK inhibitor PD98059 completely blocks insulin-stimulated increase in the Egr1 mRNA but has only a moderate effect on the Egr1 protein. At the same time, mTORC1 inhibitors rapamycin and PP242 suppress expression of the Egr1 protein and have an opposite effect on the Egr1 mRNA. Mouse embryonic fibroblasts with genetic ablations of TSC2 or 4E-BP1/2 express less Egr1 mRNA but more Egr1 protein than wild type controls. (35)S-labeling has confirmed that translation of the Egr1 mRNA is much more effective in 4E-BP1/2-null cells than in control. A selective agonist of the CB1 receptors, ACEA, up-regulates Egr1 mRNA, but does not activate mTORC1 and does not increase Egr1 protein in adipocytes. These data suggest that although insulin activates both the Erk and the mTORC1 signaling pathways in adipocytes, regulation of the Egr1 expression takes place predominantly via the mTORC1/4E-BP-mediated axis. In confirmation of this model, we show that 4E-BP1/2-null MEFs express less ATGL and accumulate more fat than control cells, while knock down of Egr1 in 4E-BP1/2-null MEFs increases ATGL expression and decreases fat storage.

Fat-specific protein 27 inhibits lipolysis by facilitating the inhibitory effect of transcription factor Egr1 on transcription of adipose triglyceride lipase.

Lipolysis in fat tissue represents a major source of circulating fatty acids. Previously, we have found that lipolysis in adipocytes is controlled by early growth response transcription factor Egr1 that directly inhibits transcription of adipose triglyceride lipase, ATGL (Chakrabarti, P., Kim, J. Y., Singh, M., Shin, Y. K., Kim, J., Kumbrink, J., Wu, Y., Lee, M. J., Kirsch, K. H., Fried, S. K., and Kandror, K. V. (2013) Mol. Cell. Biol. 33, 3659-3666). Here we demonstrate that knockdown of the lipid droplet protein FSP27 (a.k.a. CIDEC) in human adipocytes increases expression of ATGL at the level of transcription, whereas overexpression of FSP27 has the opposite effect. FSP27 suppresses the activity of the ATGL promoter in vitro, and the proximal Egr1 binding site is responsible for this effect. FSP27 co-immunoprecipitates with Egr1 and increases its association with and inhibition of the ATGL promoter. Knockdown of Egr1 attenuates the inhibitory effect of FSP27. These results provide a new model of transcriptional regulation of ATGL.

Akt may associate with insulin-responsive vesicles via interaction with sortilin.

Insulin-responsive vesicles (IRVs) deliver the glucose transporter Glut4 to the plasma membrane in response to activation of the insulin signaling cascade: insulin receptor-IRS-PI3 kinase-Akt-TBC1D4-Rab10. Previous studies have shown that Akt, TBC1D4, and Rab10 are compartmentalized on the IRVs. Although functionally significant, the mechanism of Akt association with the IRVs remains unknown. Using pull-down assays, immunofluorescence microscopy, and cross-linking, we have found that Akt may be recruited to the IRVs via the interaction with the juxtamembrane domain of the cytoplasmic C terminus of sortilin, a major IRV protein. Overexpression of full-length sortilin increases insulin-stimulated phosphorylation of TBC1D4 and glucose uptake in adipocytes, while overexpression of the cytoplasmic tail of sortilin has the opposite effect. Our findings demonstrate that the IRVs represent both a scaffold and a target of insulin signaling.

The sugar is sIRVed: sorting Glut4 and its fellow travelers.

Translocation of Glut4 to the plasma membrane of fat and skeletal muscle cells is mediated by specialized insulin-responsive vesicles (IRVs), whose protein composition consists primarily of glucose transporter isoform 4 (Glut4), insulin-responsive amino peptidase (IRAP), sortilin, lipoprotein receptor-related protein 1 (LRP1) and v-SNAREs. How can these proteins find each other in the cell and form functional vesicles after endocytosis from the plasma membrane? We are proposing a model according to which the IRV component proteins are internalized into sorting endosomes and are delivered to the IRV donor compartment(s), recycling endosomes and/or the trans-Golgi network (TGN), by cellugyrin-positive transport vesicles. The cytoplasmic tails of Glut4, IRAP, LRP1 and sortilin play an important targeting role in this process. Once these proteins arrive in the donor compartment, they interact with each other via their lumenal domains. This facilitates clustering of the IRV proteins into an oligomeric complex, which can then be distributed from the donor membranes to the IRV as a single entity with the help of adaptors, such as Golgi-localized, gamma-adaptin ear-containing, ARF-binding (GGA).

Inactivation of Minar2 in mice hyperactivates mTOR signaling and results in obesity.

OBJECTIVE: Obesity is a complex disorder and is linked to chronic diseases such as type 2 diabetes. Major intrinsically disordered NOTCH2-associated receptor2 (MINAR2) is an understudied protein with an unknown role in obesity and metabolism. The purpose of this study was to determine the impact of Minar2 on adipose tissues and obesity. METHOD: We generated Minar2 knockout (KO) mice and used various molecular, proteomic, biochemical, histopathology, and cell culture studies to determine the pathophysiological role of Minar2 in adipocytes. RESULTS: We demonstrated that the inactivation of Minar2 results in increased body fat with hypertrophic adipocytes. Minar2 KO mice on a high-fat diet develop obesity and impaired glucose tolerance and metabolism. Mechanistically, Minar2 interacts with Raptor, a specific and essential component of mammalian TOR complex 1 (mTORC1) and inhibits mTOR activation. mTOR is hyperactivated in the adipocytes deficient for Minar2 and over-expression of Minar2 in HEK-293 cells inhibited mTOR activation and phosphorylation of mTORC1 substrates, including S6 kinase, and 4E-BP1. CONCLUSION: Our findings identified Minar2 as a novel physiological negative regulator of mTORC1 with a key role in obesity and metabolic disorders. Impaired expression or activation of MINAR2 could lead to obesity and obesity-associated diseases.

An ACAP1-containing clathrin coat complex for endocytic recycling.

Whether coat proteins play a widespread role in endocytic recycling remains unclear. We find that ACAP1, a GTPase-activating protein (GAP) for ADP-ribosylation factor (ARF) 6, is part of a novel clathrin coat complex that is regulated by ARF6 for endocytic recycling in two key physiological settings, stimulation-dependent recycling of integrin that is critical for cell migration and insulin-stimulated recycling of glucose transporter type 4 (Glut4), which is required for glucose homeostasis. These findings not only advance a basic understanding of an early mechanistic step in endocytic recycling but also shed key mechanistic insights into major physiological events for which this transport plays a critical role.

SIRT1 controls lipolysis in adipocytes via FOXO1-mediated expression of ATGL.

Recent studies have established SIRT1 as an important regulator of lipid metabolism, although the mechanism of its action at the molecular level has not been revealed. Here, we show that knockdown of SIRT1 with the help of small hairpin RNA decreases basal and isoproterenol-stimulated lipolysis in cultured adipocytes. This effect is attributed, at least in part, to the suppression of the rate-limiting lipolytic enzyme, adipose triglyceride lipase (ATGL), at the level of transcription. Mechanistically, SIRT1 controls acetylation status and functional activity of FoxO1 that directly binds to the ATGL promoter and regulates ATGL gene transcription. We have also found that depletion of SIRT1 decreases AMP-dependent protein kinase (AMPK) activity in adipocytes. To determine the input of AMPK in regulation of lipolysis, we have established a stable adipose cell line that expresses a dominant-negative α1 catalytic subunit of AMPK under the control of the inducible TET-OFF lentiviral expression vector. Reduction of AMPK activity does not have a significant effect on the rates of lipolysis in this cell model. We conclude, therefore, that SIRT1 controls ATGL transcription primarily by deacetylating FoxO1.

Proteomic analysis of GLUT4 storage vesicles reveals LRP1 to be an important vesicle component and target of insulin signaling.

Insulin stimulates the translocation of intracellular GLUT4 to the plasma membrane where it functions in adipose and muscle tissue to clear glucose from circulation. The pathway and regulation of GLUT4 trafficking are complicated and incompletely understood and are likely to be contingent upon the various proteins other than GLUT4 that comprise and interact with GLUT4-containing vesicles. Moreover, not all GLUT4 intracellular pools are insulin-responsive as some represent precursor compartments, thus posing a biochemical challenge to the purification and characterization of their content. To address these issues, we immunodepleted precursor GLUT4-rich vesicles and then immunopurified GLUT4 storage vesicle (GSVs) from primary rat adipocytes and subjected them to semi-quantitative and quantitative proteomic analysis. The purified vesicles translocate to the cell surface almost completely in response to insulin, the expected behavior for bona fide GSVs. In total, over 100 proteins were identified, about 50 of which are novel in this experimental context. LRP1 (low density lipoprotein receptor-related protein 1) was identified as a major constituent of GSVs, and we show it interacts with the lumenal domains of GLUT4 and other GSV constituents. Its cytoplasmic tail interacts with the insulin-signaling pathway target, AS160 (Akt substrate of 160 kDa). Depletion of LRP1 from 3T3-L1 adipocytes reduces GLUT4 expression and correspondingly results in decreased insulin-stimulated 2-[(3)H]deoxyglucose uptake. Furthermore, adipose-specific LRP1 knock-out mice also exhibit decreased GLUT4 expression. These findings suggest LRP1 is an important component of GSVs, and its expression is needed for the formation of fully functional GSVs.

Cerebellar neurons possess a vesicular compartment structurally and functionally similar to Glut4-storage vesicles from peripheral insulin-sensitive tissues.

The insulin-sensitive isoform of the glucose transporting protein, Glut4, is expressed in fat as well as in skeletal and cardiac muscle and is responsible for the effect of insulin on blood glucose clearance. Recent studies have revealed that Glut4 is also expressed in the brain, although the intracellular compartmentalization and regulation of Glut4 in neurons remains unknown. Using sucrose gradient centrifugation, immunoadsorption and immunofluorescence staining, we have shown that Glut4 in the cerebellum is localized in intracellular vesicles that have the sedimentation coefficient, the buoyant density, and the protein composition similar to the insulin-responsive Glut4-storage vesicles from fat and skeletal muscle cells. In cultured cerebellar neurons, insulin stimulates glucose uptake and causes translocation of Glut4 to the cell surface. Using 18FDG (18fluoro-2-deoxyglucose) positron emission tomography, we found that physical exercise acutely increases glucose uptake in the cerebellum in vivo. Prolonged physical exercise increases expression of the Glut4 protein in the cerebellum. Our results suggest that neurons have a novel type of translocation-competent vesicular compartment which is regulated by insulin and physical exercise similar to Glut4-storage vesicles in peripheral insulin target tissues.

Sortilin is essential and sufficient for the formation of Glut4 storage vesicles in 3T3-L1 adipocytes.

Impaired translocation of the glucose transporter isoform 4 (Glut4) to the plasma membrane in fat and skeletal muscle cells may represent a primary defect in the development of type 2 diabetes mellitus. Glut4 is localized in specialized storage vesicles (GSVs), the biological nature and biogenesis of which are not known. Here, we report that GSVs are formed in differentiating 3T3-L1 adipocytes upon induction of sortilin on day 2 of differentiation. Forced expression of Glut4 prior to induction of sortilin leads to rapid degradation of the transporter, whereas overexpression of sortilin increases formation of GSVs and stimulates insulin-regulated glucose uptake. Knockdown of sortilin decreases both formation of GSVs and insulin-regulated glucose uptake. Finally, we have reconstituted functional GSVs in undifferentiated cells by double transfection of Glut4 and sortilin. Thus, sortilin is not only essential, but also sufficient for biogenesis of GSVs and acquisition of insulin responsiveness in adipose cells.

The C-terminus of GLUT4 targets the transporter to the perinuclear compartment but not to the insulin-responsive vesicles.

Postprandial blood glucose clearance is mediated by GLUT4 (glucose transporter 4) which is translocated from an intracellular storage pool to the plasma membrane in response to insulin. The nature of the intracellular storage pool of GLUT4 is not well understood. Immunofluorescence staining shows that, under basal conditions, the major population of GLUT4 resides in the perinuclear compartment. At the same time, biochemical fractionation reveals that GLUT4 is localized in IRVs (insulin-responsive vesicles). The relationship between the perinuclear GLUT4 compartment and the IRVs is not known. In the present study, we have exchanged the C-termini of GLUT4 and cellugyrin, another vesicular protein that is not localized in the IRVs and has no insulin response. We have found that GLUT4 with the cellugyrin C-terminus loses its specific perinuclear localization, whereas cellugyrin with the GLUT4 C-terminus acquires perinuclear localization and becomes co-localized with GLUT4. This, however, is not sufficient for the effective entry of the latter chimaera into the IRVs as only a small fraction of cellugyrin with the GLUT4 C-terminus is targeted to the IRVs and is translocated to the plasma membrane in response to insulin stimulation. We suggest that the perinuclear GLUT4 storage compartment comprises the IRVs and the donor membranes from which the IRVs originate. The C-terminus of GLUT4 is required for protein targeting to the perinuclear donor membranes, but not to the IRVs.

Nutrients suppress phosphatidylinositol 3-kinase/Akt signaling via raptor-dependent mTOR-mediated insulin receptor substrate 1 phosphorylation.

Nutritional excess and/or obesity represent well-known predisposition factors for the development of non-insulin-dependent diabetes mellitus (NIDDM). However, molecular links between obesity and NIDDM are only beginning to emerge. Here, we demonstrate that nutrients suppress phosphatidylinositol 3 (PI3)-kinase/Akt signaling via Raptor-dependent mTOR (mammalian target of rapamycin)-mediated phosphorylation of insulin receptor substrate 1 (IRS-1). Raptor directly binds to and serves as a scaffold for mTOR-mediated phosphorylation of IRS-1 on Ser636/639. These serines lie close to the Y(632)MPM motif that is implicated in the binding of p85alpha/p110alpha PI3-kinase to IRS-1 upon insulin stimulation. Phosphomimicking mutations of these serines block insulin-stimulated activation of IRS-1-associated PI3-kinase. Knockdown of Raptor as well as activators of the LKB1/AMPK pathway, such as the widely used antidiabetic compound metformin, suppress IRS-1 Ser636/639 phosphorylation and reverse mTOR-mediated inhibition on PI3-kinase/Akt signaling. Thus, diabetes-related hyperglycemia hyperactivates the mTOR pathway and may lead to insulin resistance due to suppression of IRS-1-dependent PI3-kinase/Akt signaling.

The mammalian target of rapamycin complex 1 regulates leptin biosynthesis in adipocytes at the level of translation: the role of the 5'-untranslated region in the expression of leptin messenger ribonucleic acid.

Leptin production by adipose cells in vivo is increased after feeding and decreased by food deprivation. However, molecular mechanisms that control leptin expression in response to food intake remain unknown. Here, we test the hypothesis that leptin expression in adipose cells is regulated by nutrient- and insulin-sensitive mammalian target of rapamycin complex 1 (mTORC1)-mediated pathway. The activity of mTORC1 in 3T3-L1 adipocytes was up-regulated by stable expression of either constitutively active Rheb or dominant-negative AMP-activated protein kinase. In both cases, expression of endogenous leptin was significantly elevated at the level of translation. To investigate the role of leptin 5'-untranslated region (UTR) in the regulation of protein expression, we created bicistronic reporter constructs with and without the 5'-UTR. We found that the presence of leptin 5'-UTR renders mRNA resistant to regulation by mTORC1. It appears, therefore, that mTORC1 controls translation of leptin mRNA via a novel mechanism that does not require the presence of either the 5'-terminal oligopyrimidine tract or the 5'-UTR.

Insulin-responsive amino peptidase follows the Glut4 pathway but is dispensable for the formation and translocation of insulin-responsive vesicles.

In fat and skeletal muscle cells, insulin-responsive amino peptidase (IRAP) along with glucose transporter 4 (Glut4) and sortilin, represents a major component protein of the insulin-responsive vesicles (IRVs). Here, we show that IRAP, similar to Glut4 and sortilin, is retrieved from endosomes to the trans-Golgi network by retromer. Unlike Glut4, retrograde transport of IRAP does not require sortilin, as retromer can directly bind to the cytoplasmic tail of IRAP. Ablation of IRAP in 3T3-L1 adipocytes shifts the endosomal pool of Glut4 to more acidic endosomes, but does not affect IRV targeting, stability, and insulin responsiveness of Glut4.

ATGL-1 mediates the effect of dietary restriction and the insulin/IGF-1 signaling pathway on longevity in C. elegans.

OBJECTIVE: Animal lifespan is controlled through genetic pathways that are conserved from nematodes to humans. Lifespan-promoting conditions in nematodes include fasting and a reduction of insulin/IGF signaling. Here we aimed to investigate the input of the Caenorhabditis elegans homologue of the mammalian rate-limiting lipolytic enzyme Adipose Triglyceride Lipase, ATGL-1, in longevity control. METHODS: We used a combination of genetic and biochemical approaches to determine the role of ATGL-1 in accumulation of triglycerides and regulation of longevity. RESULTS: We found that expression of ATGL is increased in the insulin receptor homologue mutant daf-2 in a FoxO/DAF-16-dependent manner. ATGL-1 is also up-regulated by fasting and in the eat-2 loss-of-function mutant strain. Overexpression of ATGL-1 increases basal and maximal oxygen consumption rate and extends lifespan in C. elegans. Reduction of ATGL-1 function suppresses longevity of the long-lived mutants eat-2 and daf-2. CONCLUSION: Our results demonstrate that ATGL is required for extended lifespan downstream of both dietary restriction and reduced insulin/IGF signaling.

Study of glucose uptake in adipose cells.

Glucose is the main metabolic fuel in mammalian cells. Glucose entry into cells is facilitated by a family of ubiquitously expressed glucose transporter proteins. Typically, glucose transporters are localized on the plasma membrane. One notable exception is the glucose transporter isoform 4 (Glut4), which is specifically expressed in insulin sensitive tissues, i.e., skeletal muscle, heart muscle, and fat, and is responsible for the insulin effect on blood glucose clearance (1). Under basal conditions, Glut4 is compartmentalized in intracellular membrane vesicles and thus has no access to the extracellular space. Upon insulin administration, Glut4-containing vesicles fuse with the plasma membrane and deliver the transporter to its site of action. As a result, Glut4 content on the plasma membrane is increased, and glucose uptake in the cell is significantly elevated. Here, we describe two complementary techniques. The first one uses tritiated 2-deoxyglucose and is designed to measure insulin-stimulated glucose transport into cultured adipose cells. The second allows one to quantify the degree of Glut4 translocation from an intracellular compartment to the plasma membrane.

Alpha-synuclein is localized in a subpopulation of rat brain synaptic vesicles.

Alpha-synuclein is a neuronal protein implicated both in synaptic transmission and in neurodegenerative diseases. Although it is evident that this protein is enriched in the presynaptic terminals of neurons, localization in synaptic vesicles has not been conclusively determined. Here, we show that alpha-synuclein is present, but not enriched, in synaptic vesicles using highly purified synaptic vesicle preparations from rat brain homogenate. Immunoisolation of vesicles using antibodies against synaptophysin or synaptobrevin confirmed the presence of alpha-synuclein in synaptic vesicles. Additional separation of synaptic vesicles by sucrose velocity centrifugation showed that there are different subpopulations of synaptic vesicles and that alpha-synuclein is present only in a specific subpopulation, whereas synaptophysin and synaptobrevin were found in all the synaptic vesicles. Presence of alpha-synuclein only in a subset of synaptic vesicles suggests that this protein may have a specific function in synaptic vesicle cycling, hence in synaptic transmission.

Detection of Detergent-sensitive Interactions Between Membrane Proteins.

Our ability to explore protein-protein interactions is the key to understanding regulatory connections in the cell. However, detection of protein-protein interactions in many cases is associated with significant experimental challenges. In particular, sorting receptors interact with their protein cargo in the lumen of the membrane compartments often in a detergent-sensitive fashion, making co-immunoprecipitation of these proteins unusable. Binding of the sorting receptor sortilin to glucose transporter GLUT4 may serve as an example of weak luminal interactions between membrane proteins. Here, we describe a fast, simple, and inexpensive assay to validate the interaction between sortilin and GLUT4. For that, we have designed and chemically synthesized the myc-tagged peptide corresponding to the potential sortilin-binding epitope in the luminal part of GLUT4. Sortilin tagged with six histidines was expressed in mammalian cells, and isolated from cell lysates using Cobalt beads. Sortilin immobilized on the beads was incubated with the peptide solution at different pH values, and the eluted material was analyzed by Western blotting. This assay can be easily adapted to study other detergent-sensitive protein-protein interactions.

Phosphatidylinositol 4-kinase type IIalpha is targeted specifically to cellugyrin-positive glucose transporter 4 vesicles.

Phosphoinositides now emerge as important regulators of membrane traffic. In particular, phosphatidylinositol 4-phosphate may serve as a precursor for polyphosphorylated derivatives of phosphatidylinositol and, also, may regulate vesicular traffic by recruiting specific proteins to the membrane. Early results have demonstrated the presence of phosphatidylinositol 4-kinase (PI4K) activity in glucose transporter 4 (Glut4) vesicles from fat and skeletal muscle cells. However, the molecular identity of phosphatidylinositol 4-kinase(s) associated with Glut4 vesicles has not been characterized. It has also been determined that Glut4 vesicles are not homogeneous and represent a mixture of at least two vesicular populations: ubiquitous cellugyrin-positive transport vesicles and specialized cellugyrin-negative insulin-responsive Glut4 storage vesicles, which are different in size, protein composition, and functional properties. Using sequential immunoadsorption, subcellular fractionation, and immunofluorescence staining, we show that virtually all PI4K activity in Glut4 vesicles is represented by PI4K type IIalpha, which is associated with cellugyrin-positive vesicles and is not detectable in the Glut4 storage vesicles. The unique N terminus of PI4K type IIalpha is required for the targeting of the enzyme to cellugyrin-positive vesicles. Knockdown of PI4K type IIalpha with the help of short hairpin RNA does not decrease the amount of cellugyrin-positive vesicles in human embryonic kidney 293 cells.