Mitogen-activated kinase kinase kinase kinase 4 (Map4k4), originally identified in small interfering (si)RNA screens and characterized by tissue-specific gene deletions, is emerging as a regulator of glucose homeostasis and cardiovascular health. Recent studies have shown that Map4k4 gene ablation or inhibition of its kinase activity attenuates hyperglycemia and plaque formation in mouse models of insulin resistance and atherosclerosis, and suggest roles for Map4k4 in multiple signaling systems, including NFκB activation, small GTPase regulation, the Hippo cascade, and regulation of cell dynamics by FERM domain proteins. This new and promising area of inquiry raises key questions that need to be addressed, such as defining which of the above or other effectors mediate Map4k4 control of metabolic and vascular functions, and identifying upstream activators of Map4k4.

Objective: Adipose tissue relies on lipid droplet (LD) proteins in its role as a lipid-storing endocrine organ that controls whole body metabolism. Hypoxia-inducible Gene 2 (Hig2) is a recently identified LD-associated protein in hepatocytes that promotes hepatic lipid storage, but its role in the adipocyte had not been investigated. Here we tested the hypothesis that Hig2 localization to LDs in adipocytes promotes adipose tissue lipid deposition and systemic glucose homeostasis.Method: White and brown adipocyte-deficient (Hig2(fl/fl) x Adiponection cre+) and selective brown/beige adipocyte-deficient (Hig2 (fl/fl) x Ucp1 cre+) mice were generated to investigate the role of Hig2 in adipose depots. Additionally, we used multiple housing temperatures to investigate the role of active brown/beige adipocytes in this process.Results: Hig2 localized to LDs in SGBS cells, a human adipocyte cell strain. Mice with adipocyte-specific Hig2 deficiency in all adipose depots demonstrated reduced visceral adipose tissue weight and increased glucose tolerance. This metabolic effect could be attributed to brown/beige adipocyte-specific Hig2 deficiency since Hig2 (fl/fl) x Ucp1 cre+ mice displayed the same phenotype. Furthermore, when adipocyte-deficient Hig2 mice were moved to thermoneutral conditions in which non-shivering thermogenesis is deactivated, these improvements were abrogated and glucose intolerance ensued. Adipocyte-specific Hig2 deficient animals displayed no detectable changes in adipocyte lipolysis or energy expenditure, suggesting that Hig2 may not mediate these metabolic effects by restraining lipolysis in adipocytes.Conclusions: We conclude that Hig2 localizes to LDs in adipocytes, promoting adipose tissue lipid deposition and that its selective deficiency in active brown/beige adipose tissue mediates improved glucose tolerance at 23 degrees C. Reversal of this phenotype at thermoneutrality in the absence of detectable changes in energy expenditure, adipose mass, or liver triglyceride suggests that Hig2 deficiency triggers a deleterious endocrine or neuroendocrine pathway emanating from brown/beige fat cells. (C) 2016 The Author(s). Published by Elsevier GmbH.

Studies in vitro suggest that mitogen-activated protein kinase kinase kinase kinase 4 (Map4k4) attenuates insulin signaling, but confirmation in vivo is lacking since Map4k4 knockout is lethal during embryogenesis. We thus generated mice with floxed Map4k4 alleles and a tamoxifen-inducible Cre/ERT2 recombinase under the control of the ubiquitin C promoter to induce whole-body Map4k4 deletion after these animals reached maturity. Tamoxifen administration to these mice induced Map4k4 deletion in all tissues examined, causing decreased fasting blood glucose concentrations and enhanced insulin signaling to AKT in adipose tissue and liver but not in skeletal muscle. Surprisingly, however, mice generated with a conditional Map4k4 deletion in adiponectin-positive adipocytes or in albumin-positive hepatocytes displayed no detectable metabolic phenotypes. Instead, mice with Map4k4 deleted in Myf5-positive tissues, including all skeletal muscles tested, were protected from obesity-induced glucose intolerance and insulin resistance. Remarkably, these mice also showed increased insulin sensitivity in adipose tissue but not skeletal muscle, similar to the metabolic phenotypes observed in inducible whole-body knockout mice. Taken together, these results indicate that (i) Map4k4 controls a pathway in Myf5-positive cells that suppresses whole-body insulin sensitivity and (ii) Map4k4 is a potential therapeutic target for improving glucose tolerance and insulin sensitivity in type 2 diabetes.

Phosphatidylinositol-3-phosphate (PtdIns(3)P) is one of several bioactive phosphorylated products of the action of PI 3-kinases. PtdIns(3)P is localized primarily in the endomembrane systems of eukaryotic cells and is recognized by proteins with one of several recently identified PtdIns(3)P-binding domains. PtdIns(3)P and these associated proteins play roles in cellular functions associated with the endomembrane system, including endocytosis, phagocytosis, growth factor signaling, and autophagy.

Phagocytic macrophages and dendritic cells are desirable targets for potential RNAi (RNA interference) therapeutics because they often mediate pathogenic inflammation and autoimmune responses. We recently engineered a complex 5 component glucan-based encapsulation system for siRNA (small interfering RNA) delivery to phagocytes. In experiments designed to simplify this original formulation, we discovered that the amphipathic peptide Endo-Porter forms stable nanocomplexes with siRNA that can mediate potent gene silencing in multiple cell types. In order to restrict such gene silencing to phagocytes, a method was developed to entrap siRNA-Endo-Porter complexes in glucan shells of 2-4 μm diameter in the absence of other components. The resulting glucan particles containing fluorescently labelled siRNA were readily internalized by macrophages, but not other cell types, and released the labelled siRNA into the macrophage cytoplasm. Intraperitoneal administration of such glucan particles containing siRNA-Endo-Porter complexes to mice caused gene silencing specifically in macrophages that internalized the particles. These results from the present study indicate that specific targeting to phagocytes is mediated by the glucan, whereas Endo-Porter peptide serves both to anchor siRNA within glucan particles and to catalyse escape of siRNA from phagosomes. Thus we have developed a simplified siRNA delivery system that effectively and specifically targets phagocytes in culture or in intact mice.

Considerable evidence has implicated the involvement of phosphatidylinositol (PI) 3-kinase in the insulin-stimulated translocation of the GLUT4 glucose transporter to the surface of fat cells [reviewed in 1]. The observation of an increase in Pl 3,4-bisphosphate and Pl 3,4.5 triphosphate in insulin-stimulated cells, the association of the p85/pl 10 P13kinase with IRS-i as well as evidence of P13-kinase translocation to GLUT4-enriched membranes in response to insulin all point to a critical role of P13-kinase in insulin action. Furthermore, wortmannin, a potent inhibitor of some P13kinase activities, abrogates the insulin-stimulated increase in cell surface GLUT4. The well characterized pl 10 P13-kinase is likely a major contributor to this insulin regulated P13-kinase activity since this enzyme is capable of producing the growth factor-regulated phosphoinositides Pl 3,4-P2 and PI 3,4,5-P3, and is sensitive to the inhibitor wortmannm at low nanomolar concentrations. However, biochemical and genetic evidence of other P13-kinase activities in mammalian cells prompted us to search for additional P13kinase species present in insulin-responsive cells that might contribute to insulin-activated P13-kinase activity or the GLUT4 trafficking pathway. To this end, we carried out a search for P13-kinase cDNAs based on polymerase chain reaction amplification of sequences present in eDNA from fully differentiated 3T3-LI adipocytes. Primers were designed based on sequences conserved in the several known species of P13-kinase. This screen resulted in the identification of four species of P13-kinase expressed in cultured mouse adipocytes (Fig. 1). Two of these correspond to the pl 10 and pl l03 isoforms which exist in a complex with the p8S regulatory subunit. Two additional species, although clearly related to the pl 10 isoforms, were found to be novel species. Full length cDNAs encoding these proteins were isolated and sequenced. One of these was found to be most closely related to the yeast VPS34 P13-kinase and likely represents the

Acquired resistance to the action of insulin to stimulate glucose transport in skeletal muscle is associated with obesity and promotes the development of type 2 diabetes. In skeletal muscle, insulin resistance can result from high levels of circulating fatty acids that disrupt insulin signalling pathways. However, the severity of insulin resistance varies greatly among obese people. Here we postulate that this variability might reflect differences in levels of lipid-droplet proteins that promote the sequestration of fatty acids within adipocytes in the form of triglycerides, thereby lowering exposure of skeletal muscle to the inhibitory effects of fatty acids.

Adipose tissue modulates whole body metabolism and insulin sensitivity by controlling circulating lipid levels and producing molecules that can regulate fatty acid metabolism in such tissues as muscle and liver. We have developed RNA interference (RNAi) screens to identify genes in cultured adipocytes that regulate insulin signalling and key metabolic pathways. These short interfering RNA (siRNA)-based screens identified the transcriptional corepressor receptor interacting protein 140 (RIP140) (J Clin Invest 116: 125, 2006) and the mitogen-activated protein kinase (MAP4k4) (Proc Natl Acad Sci USA 103: 2087, 2006) as negative regulators of insulin-responsive hexose uptake and oxidative metabolism. Gene expression profiling revealed that RIP140 depletion upregulates the expression of clusters of genes in the pathways of glucose uptake, glycolysis, tricarboxylic acid cycle, fatty acid oxidation, mitochondrial biogenesis and oxidative phosphorylation. RIP140-null mice resist weight gain on a high-fat diet and display enhanced glucose tolerance. MAP4k4 depletion in adipocytes increases many of the RIP140-sensitive genes, increases adipogenesis and mediates some actions of tumour necrosis factor-alpha (TNF-alpha). Remarkably, another hit in our RNAi screens was fat specific protein 27 (FSP27), a highly expressed isoform of Cidea. We discovered that FSP27 unexpectedly associates specifically with lipid droplets and regulates fat storage. We conclude that RIP140, MAP4k4 and the novel lipid droplet protein FSP27 are powerful regulators of adipose tissue metabolism and are potential therapeutic targets for controlling metabolic disease. The discovery of these novel proteins validates the power of RNAi screening for discovery of new therapeutic approaches to type 2 diabetes and obesity.

Cultured adipocyte cell lines are a model system widely used to study adipose function, but they exhibit significant physiological differences compared with primary cells from adipose tissue. Here we report short interfering RNA-based methodology to selectively attenuate gene expression in mouse and human primary adipose tissues as a means of rapidly validating findings made in cultured adipocyte cell lines. The method is exemplified by depletion of the PTEN phosphatase in white adipose tissue (WAT) from mouse and humans, which increases Akt phosphorylation as expected. This technology is also shown to silence genes in mouse brown adipose tissue. Previous work revealed upregulation of the mitochondrial protein UCP1 in adipose cells from mice lacking the gene for the transcriptional corepressor RIP140, whereas in cultured adipocytes, loss of RIP140 has a little effect on UCP1 expression. Application of our method to deplete RIP140 in primary mouse WAT elicited markedly increased oxygen consumption and expression of UCP1 that exactly mimics the phenotype observed in RIP140-null mice. This ex-vivo method of gene silencing should be useful in rapid validation studies as well as in addressing the depot- and species-specific functions of genes in adipose biology.

Phosphatidylinositide (PtdIns) 3-kinase catalyzes the addition of a phosphate group to the 3′-position of phosphatidyl inositol. Accumulated evidence shows that PtdIns 3-kinase can provide a critical signal for cell proliferation, cell survival, membrane trafficking, glucose transport, and membrane ruffling. Mammalian PtdIns 3-kinases are divided into three classes based on structure and substrate specificity. A unique characteristic of class II PtdIns 3-kinases is the presence of both a phox homolog domain and a C2 domain at the C terminus. The biological function of the C2 domain of the class II PtdIns 3-kinases remains to be determined. We have determined the crystal structure of the mCPK-C2 domain, which is the first three-dimensional structural model of a C2 domain of class II PtdIns 3-kinases. Structural studies reveal that the mCPK-C2 domain has a typical anti-parallel β-sandwich fold. Scrutiny of the surface of this C2 domain has identified three small, shallow sulfate-binding sites. On the basis of the structural features of these sulfate-binding sites, we have studied the lipid binding properties of the mCPK-C2 domain by site-directed mutagenesis. Our results show that this C2 domain binds specifically to PtdIns(3,4)P2 and PtdIns(4,5)P2 and that three lysine residues at SBS I site, Lys-1420, Lys-1432, and Lys-1434, are responsible for the phospholipid binding affinity.

The insulin-regulated glucose transporter GLUT4 is a key modulator of whole body glucose homeostasis, and its selective loss in adipose tissue or skeletal muscle causes insulin resistance and diabetes. Here we report an RNA interference-based screen of protein kinases expressed in adipocytes and identify four negative regulators of insulin-responsive glucose transport: the protein kinases PCTAIRE-1 (PCTK1), PFTAIRE-1 (PFTK1), IκB kinase α, and MAP4K4/NIK. Integrin-linked protein kinase was identified as a positive regulator of this process. We characterized one of these hits, MAP4K4/NIK, and found that it is unique among mitogen-activated protein (MAP) kinases expressed in cultured adipocytes in attenuating hexose transport. Remarkably, MAP4K4/NIK suppresses expression of the adipogenic transcription factors C/EBPα, C/EBPβ, and PPARγ and of GLUT4 itself in these cells. RNA interference-mediated depletion of MAP4K4/NIK early in differentiation enhances adipogenesis and triglyceride deposition, and even in fully differentiated adipocytes its loss up-regulates GLUT4. Conversely, conditions that inhibit adipogenesis such as TNF-α treatment or depletion of PPARγ markedly up-regulate MAP4K4/NIK expression in cultured adipocytes. Furthermore, TNF-α signaling to down-regulate GLUT4 is impaired in the absence of MAP4K4/NIK, indicating that MAP4K4 expression is required for optimal TNF-α action. These results reveal a MAP4K4/NIK-dependent signaling pathway that potently inhibits PPARγ-responsive gene expression, adipogenesis, and insulin-stimulated glucose transport.

Using an siRNA-based screen, we identified the transcriptional corepressor RIP140 as a negative regulator of insulin-responsive hexose uptake and oxidative metabolism in 3T3-L1 adipocytes. Affymetrix GeneChip profiling revealed that RIP140 depletion upregulates the expression of clusters of genes in the pathways of glucose uptake, glycolysis, TCA cycle, fatty acid oxidation, mitochondrial biogenesis, and oxidative phosphorylation in these cells. Conversely, we show that reexpression of RIP140 in mouse embryonic fibroblasts derived from RIP140-null mice downregulates expression of many of these same genes. Consistent with these microarray data, RIP140 gene silencing in cultured adipocytes increased both conversion of [14C]glucose to CO2 and mitochondrial oxygen consumption. RIP140-null mice, previously reported to resist weight gain on a high-fat diet, are shown here to display enhanced glucose tolerance and enhanced responsiveness to insulin compared with matched wild-type mice upon high-fat feeding. Mechanistically, RIP140 was found to require the nuclear receptor ERRalpha to regulate hexose uptake and mitochondrial proteins SDHB and CoxVb, although it likely acts through other nuclear receptors as well. We conclude that RIP140 is a major suppressor of adipocyte oxidative metabolism and mitochondrial biogenesis, as well as a negative regulator of whole-body glucose tolerance and energy expenditure in mice.

Using siRNA-mediated gene silencing in cultured adipocytes, we have dissected the insulin-signalling pathway leading to translocation of GLUT4 glucose transporters to the plasma membrane. RNAi (RNA interference)-based depletion of components in the putative TC10 pathway (CAP, CrkII and c-Cbl plus Cbl-b) or the phospholipase Cγ pathway failed to diminish insulin signalling to GLUT4. Within the phosphoinositide 3-kinase pathway, loss of the 5′-phosphatidylinositol 3,4,5-trisphosphate phosphatase SHIP2 was also without effect, whereas depletion of the 3′-phosphatase PTEN significantly enhanced insulin action. Downstream of phosphatidylinositol 3,4,5-trisphosphate and PDK1, silencing the genes encoding the protein kinases Akt1/PKBα, or CISK(SGK3) or protein kinases Cλ/ζ had little or no effect, but loss of Akt2/PKBβ significantly attenuated GLUT4 regulation by insulin. These results show that Akt2/PKBβ is the key downstream intermediate within the phosphoinositide 3-kinase pathway linked to insulin action on GLUT4 in cultured adipocytes, whereas PTEN is a potent negative regulator of this pathway.

Ca2+ is an essential requirement in membrane fusion, acting through binding proteins such as calmodulin (CaM). Ca2+/CaM is required for early endosome fusion in vitro, however, the molecular basis for this requirement is unknown. An additional requirement for endosome fusion is the protein Early Endosome Antigen 1 (EEA1), and its recruitment to the endosome depends on phosphatidylinositol 3-phosphate [PI(3)P] and the Rab5 GTPase. Herein, we demonstrate that inhibition of Ca2+/CaM, by using either chemical inhibitors or specific antibodies directed to CaM, results in a profound inhibition of EEA1 binding to endosomal membranes both in live cells and in vitro. The concentration of Ca2+/CaM inhibitors required for a full dissociation of EEA1 from endosomal membranes had no effect on the activity of phosphatidylinositol 3-kinases or on endogenous levels of PI(3)P. However, the interaction of EEA1 with liposomes containing PI(3)P was decreased by Ca2+/CaM inhibitors. Thus, Ca2+/CaM seems to be required for the stable interaction of EEA1 with endosomal PI(3)P, perhaps by directly or indirectly stabilizing the quaternary organization of the C-terminal FYVE domain of EEA1. This requirement is likely to underlie at least in part the essential role of Ca2+/CaM in endosome fusion.

The Phox homology (PX) domain has recently been reported to bind to phosphoinositides, and some PX domains can localize to endosomes in vivo. Here we show data to support the conclusion that the p40(phox) PX domain binds to phosphatidylinositol 3-phosphate specifically in vitro and localizes to endosomes in intact cells. In addition, its Y59A/L65Q mutant, which has decreased affinity for phosphatidylinositol 3-phosphate in vitro, fails to target EGFP-p40-PX to endosomes. However, unlike published results, we find that the p47(phox) PX domain weakly binds to many phosphoinositides in vitro showing slightly higher affinity for phosphatidylinositol 3,4,5-trisphosphate. Moreover, we show for the first time that upon insulin-like growth factor-1 stimulation of COS cells, the p47(phox) PX domain is localized to the plasma membrane, and this subcellular localization is dependent on PI 3-kinase activity. Unexpectedly, its R42Q mutant that loses in vitro phosphoinositide-binding ability can still target EGFP-p47-PX to the plasma membrane. Our data suggest that the translocation of p47(phox) PX domain to the plasma membrane does involve 3'-phosphoinositide(s) in the process, but the phosphoinositide-binding of p47(phox) PX domain is not sufficient to recruit it to the plasma membrane. Therefore, the p40(phox) and p47(phox) PX domains can target subcellular membranes via direct or indirect recruitment by phosphoinositides, while both are under the control of phosphatidylinositol 3-kinase activity.

Protein kinases of the Akt and related serum- and glucocorticoid-regulated kinase (SGK) families are major downstream mediators of phosphatidylinositol (PI) 3-kinase signaling to many cellular processes including metabolic flux, membrane trafficking, and apoptosis. Activation of these kinases is thought to occur at the plasma membrane through their serine and threonine phosphorylation by the phosphoinositide-dependent kinase 1 (PDK1) protein kinase, which interacts with membrane 3′-polyphosphoinositides through its pleckstrin homology (PH) domain. Here, we demonstrate that the SGK family member cytokine-independent survival kinase (CISK) binds strongly and selectively to the monophosphoinositide PI(3)P through its phox homology (PX) domain. Comparing native green fluorescent protein-CISK (EGFP-CISK) to a mutant EGFP-CISK (Y51A) that displays attenuated binding to PI(3)P reveals that this interaction is both necessary and sufficient for its localization to early endosome antigen (EEA1)-positive endosomes. Furthermore, early endosome association of expressed epitope-tagged CISK in COS cells directed by binding of its PX domain to PI(3)P is required for activation of the CISK protein kinase by both insulin-like growth factor-1 and epidermal growth factor. Taken together, these results reveal a critical role of endosomal PI(3)P in the signal transmission mechanism whereby this survival kinase is activated in response to PI3-kinase stimulation by growth factors.

The recruitment of specific cytosolic proteins to intracellular membranes through binding phosphorylated derivatives of phosphatidylinositol (PtdIns) controls such processes as endocytosis, regulated exocytosis, cytoskeletal organization, and cell signaling. Protein modules such as FVYE domains and PH domains that bind specifically to PtdIns 3-phosphate (PtdIns-3-P) and polyphosphoinositides, respectively, can direct such membrane targeting. Here we show that two representative Phox homology (PX) domains selectively bind to specific phosphatidylinositol phosphates. The PX domain of Vam7p selectively binds PtdIns-3-P, while the PX domain of the CPK PI-3 kinase selectively binds PtdIns-4,5-P(2). In contrast, the PX domain of Vps5p displays no binding to any PtdInsPs that were tested. In addition, the double mutant (Y42A/L48Q) of the PX domain of Vam7p, reported to cause vacuolar trafficking defects in yeast, has a dramatically decreased level of binding to PtdIns-3-P. These data reveal that the membrane targeting function of the Vam7p PX domain is based on its ability to associate with PtdIns-3-P, analogous to the function of FYVE domains.

Treating cells with the phosphatidylinositol-3-OH kinase (PI(3)K) inhibitor wortmannin causes the dissociation of the early-endosomal antigen EEA1 from early endosomes 1 . EEA1 from cytosolic extracts binds to liposomes containing phosphatidylinositol-3-phosphate (PtdIns(3)P), the major product of PI(3)K in yeast and mammalian cells 1 , 2 . Here we show that a RING zinc-finger domain at the carboxy terminus of EEA1, previously identified and named the ‘FYVE’ domain 3 , binds directly and specifically to PtdIns(3)P. This indicates that proteins containing this motif may be downstream effectors of PI(3)K in yeast and mammalian cells.

Signal transmission by many cell surface receptors results in the activation of phosphoinositide (PI) 3-kinases that phosphorylate the 3′ position of polyphosphoinositides. From a screen for mouse proteins that bind phosphoinositides, the protein GRP1 was identified. GRP1 binds phosphatidylinositol-3,4,5-trisphosphate [PtdIns(3,4,5)P 3 ] through a pleckstrin homology (PH) domain and displays a region of high sequence similarity to the yeast Sec7 protein. The PH domain of the closely related protein cytohesin-1, which, through its Sec7 homology domain, regulates integrin β2 and catalyzes guanine nucleotide exchange of the small guanine nucleotide-binding protein ARF1, was also found to specifically bind PtdIns(3,4,5)P 3 . GRP1 and cytohesin-1 appear to connect receptor-activated PI 3-kinase signaling pathways with proteins that mediate biological responses such as cell adhesion and membrane trafficking.