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.

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.

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.

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.

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.

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.

Mammalian target of rapamycin complex 1 and FoxO1 in the transcriptional control of lipolysis and de novo lipogenesis.

PURPOSE OF REVIEW: Postprandial suppression of lipolysis in adipose tissue and stimulation of de novo lipogenesis (DNL) in the liver by insulin are essential for the metabolic homeostasis in the mammalian organism. The mechanism of coregulation of lipolysis and DNL is not clear. RECENT FINDINGS: Insulin controls both lipolysis and DNL at the level of transcription via the same mammalian target of rapamycin complex 1 (mTORC1) and FoxO1-mediated signaling pathways. SUMMARY: mTORC1 suppresses lipolysis in adipose tissue and activates DNL in the liver, whereas FoxO1 has the opposite effect. Individual inputs of either mTORC1 or FoxO1 in the regulation of lipid metabolism may be difficult to evaluate because of the cross talk between these pathways.

Sortilin and retromer mediate retrograde transport of Glut4 in 3T3-L1 adipocytes.

Sortilin is a multiligand sorting receptor responsible for the anterograde transport of lysosomal enzymes and substrates. Here we demonstrate that sortilin is also involved in retrograde protein traffic. In cultured 3T3-L1 adipocytes, sortilin together with retromer rescues Glut4 from degradation in lysosomes and retrieves it to the TGN, where insulin--responsive vesicles are formed. Mechanistically, the luminal Vps10p domain of sortilin interacts with the first luminal loop of Glut4, and the cytoplasmic tail of sortilin binds to retromer. Ablation of the retromer does not affect insulin signaling but decreases the stability of sortilin and Glut4 and blocks their entry into the small vesicular carriers. As a result, Glut4 cannot reach the insulin-responsive compartment, and insulin-stimulated glucose uptake in adipocytes is suppressed. We suggest that sortilin- and retromer-mediated Glut4 retrieval from endosomes may represent a step in the Glut4 pathway vulnerable to the development of insulin resistance and diabetes.

The role of mTOR in lipid homeostasis and diabetes progression.

PURPOSE OF REVIEW: Metabolic diseases, such as type 2 diabetes, cardiac dysfunction, hypertension, and hepatic steatosis, share one critical causative factor: abnormal lipid partitioning, that redistribution of triglycerides from adipocytes to nonadipose peripheral tissues. Lipid overload of these tissues causes a number of pathological effects collectively known as lipotoxicity. If we find the way to correct lipid partitioning, we will restrain metabolic diseases, improve life quality and life expectancy and radically reduce healthcare costs. RECENT FINDINGS: Lipid partitioning in the body is maintained by tightly regulated and balanced rates of de novo lipogenesis, lipolysis, adipogenesis, and mitochondrial oxidation primarily in fat and liver. Recent studies highlighted in this review have established mTOR as a central regulator of lipid storage and metabolism. SUMMARY: Increased activity of mTOR in obesity may compensate for the negative consequences of overnutrition, whereas dysregulation of mTOR may lead to abnormal lipid partitioning and metabolic disease.

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.

Insulin responsiveness of glucose transporter 4 in 3T3-L1 cells depends on the presence of sortilin.

Insulin-dependent translocation of glucose transporter 4 (Glut4) to the plasma membrane of fat and skeletal muscle cells plays the key role in postprandial clearance of blood glucose. Glut4 represents the major cell-specific component of the insulin-responsive vesicles (IRVs). It is not clear, however, whether the presence of Glut4 in the IRVs is essential for their ability to respond to insulin stimulation. We prepared two lines of 3T3-L1 cells with low and high expression of myc7-Glut4 and studied its translocation to the plasma membrane upon insulin stimulation, using fluorescence-assisted cell sorting and cell surface biotinylation. In undifferentiated 3T3-L1 preadipocytes, translocation of myc7-Glut4 was low regardless of its expression levels. Coexpression of sortilin increased targeting of myc7-Glut4 to the IRVs, and its insulin responsiveness rose to the maximal levels observed in fully differentiated adipocytes. Sortilin ectopically expressed in undifferentiated cells was translocated to the plasma membrane regardless of the presence or absence of myc7-Glut4. AS160/TBC1D4 is expressed at low levels in preadipocytes but is induced in differentiation and provides an additional mechanism for the intracellular retention and insulin-stimulated release of Glut4.

Insulin inhibits lipolysis in adipocytes via the evolutionarily conserved mTORC1-Egr1-ATGL-mediated pathway.

One of the basic functions of insulin in the body is to inhibit lipolysis in adipocytes. Recently, we have found that insulin inhibits lipolysis and promotes triglyceride storage by decreasing transcription of adipose triglyceride lipase via the mTORC1-mediated pathway (P. Chakrabarti et al., Diabetes 59:775-781, 2010), although the mechanism of this effect remained unknown. Here, we used a genetic screen in Saccharomyces cerevisiae in order to identify a transcription factor that mediates the effect of Tor1 on the expression of the ATGL ortholog in yeast. This factor, Msn4p, has homologues in mammalian cells that form a family of early growth response transcription factors. One member of the family, Egr1, is induced by insulin and nutrients and directly inhibits activity of the ATGL promoter in vitro and expression of ATGL in cultured adipocytes. Feeding animals a high-fat diet increases the activity of mTORC1 and the expression of Egr1 while decreasing ATGL levels in epididymal fat. We suggest that the evolutionarily conserved mTORC1-Egr1-ATGL regulatory pathway represents an important component of the antilipolytic effect of insulin in the mammalian organism.

The first luminal loop confers insulin responsiveness to glucose transporter 4.

Glucose transporter isoform 4 (GLUT4), is the sole glucose transporter responsible for the effect of insulin on postprandial blood glucose clearance. However, the nature of the insulin sensitivity of GLUT4 remains unknown. In this study, we replaced the first luminal loop of cellugyrin, a 4-transmembrane protein that does not respond to insulin, with that of GLUT4. The chimera protein is targeted to the intracellular insulin-responsive vesicles and is translocated to the plasma membrane upon insulin stimulation. The faithful targeting of the chimera depends on the expression of the sorting receptor sortilin, which interacts with the unique amino acid residues in the first luminal loop of GLUT4. Thus the first luminal loop may confer insulin responsiveness to the GLUT4 molecule.

The multi-level action of fatty acids on adiponectin production by fat cells.

Current epidemics of diabetes mellitus is largely caused by wide spread obesity. The best-established connection between obesity and insulin resistance is the elevated and/or dysregulated levels of circulating free fatty acids that cause and aggravate insulin resistance, type 2 diabetes, cardiovascular disease and other hazardous metabolic conditions. Here, we investigated the effect of a major dietary saturated fatty acid, palmitate, on the insulin-sensitizing adipokine adiponectin produced by cultured adipocytes. We have found that palmitate rapidly inhibits transcription of the adiponectin gene and the release of adiponectin from adipocytes. Adiponectin gene expression is controlled primarily by PPARγ and C/EBPα. Using mouse embryonic fibroblasts from C/EBPα-null mice, we have determined that the latter transcription factor may not solely mediate the inhibitory effect of palmitate on adiponectin transcription leaving PPARγ as a likely target of palmitate. In agreement with this model, palmitate increases phosphorylation of PPARγ on Ser273, and substitution of PPARγ for the unphosphorylated mutant Ser273Ala blocks the effect of palmitate on adiponectin transcription. The inhibitory effect of palmitate on adiponectin gene expression requires its intracellular metabolism via the acyl-CoA synthetase 1-mediated pathway. In addition, we found that palmitate stimulates degradation of intracellular adiponectin by lysosomes, and the lysosomal inhibitor, chloroquine, suppressed the effect of palmitate on adiponectin release from adipocytes. We present evidence suggesting that the intracellular sorting receptor, sortilin, plays an important role in targeting of adiponectin to lysosomes. Thus, palmitate not only decreases adiponectin expression at the level of transcription but may also stimulate lysosomal degradation of newly synthesized adiponectin.

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.

Adipose triglyceride lipase: a new target in the regulation of lipolysis by insulin.

In adipose tissue, the primary physiological function of insulin is the suppression of lipolysis, the hydrolysis of stored fat. Mechanistically, insulin suppresses lipolysis both in transcriptional and post-transcriptional levels. Insulin signaling acutely inhibits beta-adrenergic signaling by decreasing intracellular cyclic AMP levels and the rate of lipolysis. Insulin also suppresses lipolysis by down-regulating the expression of the rate-limiting lipolytic enzyme, adipose triglyceride lipase or ATGL. In insulin resistance and type 2 diabetes, insulin mediated attenuation of lipolysis is impaired leading to an increased rate of lipolysis and increased release of free fatty acids (FFA) in the circulation. This is one of the potential mechanisms behind the development of hyperlipidemia and subsequent metabolic abnormalities in type 2 diabetes. In this article, we focus on the recent findings that highlight distinct molecular mechanisms by which insulin action is mediated and possible implications of the deregulation of these pathways in the pathophysiological context.

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).

Biogenesis and regulation of insulin-responsive vesicles containing GLUT4.

Insulin regulates the trafficking of GLUT4 glucose transporters in fat and muscle cells. In unstimulated cells, GLUT4 is sequestered intracellularly in small, insulin-responsive vesicles. Insulin stimulates the translocation of these vesicles to the cell surface, inserting the transporters into the plasma membrane to enhance glucose uptake. Formation of the insulin-responsive vesicles requires multiple interactions among GLUT4, IRAP, LRP1, and sortilin, as well as recruitment of GGA and ACAP1 adaptors and clathrin. Once formed, the vesicles are retained within unstimulated cells by the action of TUG, Ubc9, and other proteins. In addition to acting at other steps in vesicle recycling, insulin releases this retention mechanism to promote the translocation and fusion of the vesicles at the cell surface.

Mammalian target of rapamycin complex 1 suppresses lipolysis, stimulates lipogenesis, and promotes fat storage.

OBJECTIVE: In metazoans, target of rapamycin complex 1 (TORC1) plays the key role in nutrient- and hormone-dependent control of metabolism. However, the role of TORC1 in regulation of triglyceride storage and metabolism remains largely unknown. RESEARCH DESIGN AND METHODS: In this study, we analyzed the effect of activation and inhibition of the mammalian TORC1 (mTORC1) signaling pathway on the expression of adipose triglyceride lipase (ATGL), hormone-sensitive lipase (HSL), lipolysis, lipogenesis, and lipid storage in different mammalian cells. RESULTS: Activation of mTORC1 signaling in 3T3-L1 adipocytes by ectopic expression of Rheb inhibits expression of ATGL and HSL at the level of transcription, suppresses lipolysis, increases de novo lipogenesis, and promotes intracellular accumulation of triglycerides. Inhibition of mTORC1 signaling by rapamycin or by knockdown of raptor stimulates lipolysis primarily via activation of ATGL expression. Analogous results have been obtained in C2C12 myoblasts and mouse embryonic fibroblasts with genetic ablation of tuberous sclerosis 2 (TSC2) gene. Overexpression of ATGL in these cells antagonized the lipogenic effect of TSC2 knockout. CONCLUSIONS: Our findings demonstrate that mTORC1 promotes fat storage in mammalian cells by suppression of lipolysis and stimulation of de novo lipogenesis.