Susanna R. Keller

Cloning and characterization of subunits of the T-cell receptor and murine leukemia virus enhancer core-binding factor.

Moloney murine leukemia virus causes thymic leukemias when injected into newborn mice. A major determinant of the thymic disease specificity of Moloney virus genetically maps to the conserved viral core motif in the Moloney virus enhancer. Point mutations introduced into the core site significantly shifted the disease specificity of the Moloney virus from thymic leukemia to erythroid leukemia (N.A. Speck, B. Renjifo, E. Golemis, T.N. Fredrickson, J.W. Hartley, and N. Hopkins, Genes Dev. 4:233-242, 1990). We previously reported the purification of core-binding factors (CBF) from calf thymus nuclei (S. Wang and N.A. Speck, Mol. Cell. Biol. 12:89-102, 1992). CBF binds to core sites in murine leukemia virus and T-cell receptor enhancers. Affinity-purified CBF contains multiple polypeptides. In this study, we sequenced five tryptic peptides from two of the bovine CBF proteins and isolated three cDNA clones from a mouse thymus cDNA library encoding three of the tryptic peptides from the bovine proteins. The cDNA clones, which we call CBF beta p22.0, CBF beta p21.5, and CBF beta p17.6, encode three highly related but distinct proteins with deduced molecular sizes of 22.0, 21.5, and 17.6 kDa that appear to be translated from multiply spliced mRNAs transcribed from the same gene. CBF beta p22.0, CBF beta p21.5, and CBF beta p17.6 do not by themselves bind the core site. However, CBF beta p22.0 and CBF beta p21.5 form a complex with DNA-binding CBF alpha subunits and as a result decrease the rate of dissociation of the CBF protein-DNA complex. Association of the CBF beta subunits does not extend the phosphate contacts in the binding site. We propose that CBF beta is a non-DNA-binding subunit of CBF and does not contact DNA directly.

A Novel 160-kDa Phosphotyrosine Protein in Insulin-treated Embryonic Kidney Cells Is a New Member of the Insulin Receptor Substrate Family

We have previously identified a 160-kDa protein in human embryonic kidney (HEK) 293 cells that undergoes rapid tyrosine phosphorylation in response to insulin (PY160) (Kuhné, M. R., Zhao, Z., and Lienhard, G. E. (1995) Biochem. Biophys. Res. Commun. 211, 190–197). The phosphotyrosine form of PY160 was purified from insulin-treated HEK 293 cells by anti-phosphotyrosine immunoaffinity chromatography, the sequences of peptides determined, and its cDNA cloned. The PY160 cDNA encodes a 1257-amino acid protein that contains, in order from its N terminus, a pleckstrin homology (PH) domain, a phosphotyrosine binding (PTB) domain, and, spread over the C-terminal portion, 12 potential tyrosine phosphorylation sites. Several of these sites are in motifs expected to bind specific SH2 domain-containing proteins: YXXM (7 sites), phosphatidylinositol 3-kinase; YVNM (1 site), Grb-2; and YIEV (1 site), either the protein-tyrosine phosphatase SHP-2 or phospholipase Cγ. Furthermore, the PH and PTB domains are highly homologous (at least 40% identical) to those found in insulin receptor substrates 1, 2, and 3 (IRS-1, IRS-2, and IRS-3). Thus, PY160 is a new member of the IRS family, which we have designated IRS-4.

Cloning and Characterization of a Novel Insulin-regulated Membrane Aminopeptidase from Glut4 Vesicles

The insulin-regulated glucose transporter isotype GluT4 expressed only in muscle and adipose cells is sequestered in a specific secretory vesicle. These vesicles harbor another major protein, referred to as vp165 (for vesicle protein of 165 kDa), that like GluT4 redistributes to the plasma membrane in response to insulin. We describe here the cloning of vp165 and show that it is a novel member of the family of zinc-dependent membrane aminopeptidases, with the typical large extracellular catalytic domain and single transmembrane domain but with a unique extended cytoplasmic domain. The latter contains two dileucine motifs, which may be critical for the specific trafficking of vp165, since this has been shown to be the case for this motif in GluT4. However, the tissue distribution of vp165 is much wider than that of GluT4; consequently, vp165 may also function in processes unrelated to insulin action and may serve as a ubiquitous marker for a specialized regulated secretory vesicle.

Fat Cell–Specific Ablation of Rictor in Mice Impairs Insulin-Regulated Fat Cell and Whole-Body Glucose and Lipid Metabolism

OBJECTIVE Rictor is an essential component of mammalian target of rapamycin (mTOR) complex (mTORC) 2, a kinase that phosphorylates and activates Akt, an insulin signaling intermediary that regulates glucose and lipid metabolism in adipose tissue, skeletal muscle, and liver. To determine the physiological role of rictor/mTORC2 in insulin signaling and action in fat cells, we developed fat cell–specific rictor knockout (FRic−/−) mice. RESEARCH DESIGN AND METHODS Insulin signaling and glucose and lipid metabolism were studied in FRic−/− fat cells. In vivo glucose metabolism was evaluated by hyperinsulinemic-euglycemic clamp. RESULTS Loss of rictor in fat cells prevents insulin-stimulated phosphorylation of Akt at S473, which, in turn, impairs the phosphorylation of downstream targets such as FoxO3a at T32 and AS160 at T642. However, glycogen synthase kinase-3β phosphorylation at S9 is not affected. The signaling defects in FRic−/− fat cells lead to impaired insulin-stimulated GLUT4 translocation to the plasma membrane and decreased glucose transport. Furthermore, rictor-null fat cells are unable to suppress lipolysis in response to insulin, leading to elevated circulating free fatty acids and glycerol. These metabolic perturbations are likely to account for defects observed at the whole-body level of FRic−/− mice, including glucose intolerance, marked hyperinsulinemia, insulin resistance in skeletal muscle and liver, and hepatic steatosis. CONCLUSIONS Rictor/mTORC2 in fat cells plays an important role in whole-body energy homeostasis by mediating signaling necessary for the regulation of glucose and lipid metabolism in fat cells.

Loss of Nocturnin, a circadian deadenylase, confers resistance to hepatic steatosis and diet-induced obesity

The mammalian circadian system consists of a central oscillator in the suprachiasmatic nucleus of the hypothalamus, which coordinates peripheral clocks in organs throughout the body. Although circadian clocks control the rhythmic expression of a large number of genes involved in metabolism and other aspects of circadian physiology, the consequences of genetic disruption of circadian-controlled pathways remain poorly defined. Here we report that the targeted disruption of Nocturnin (Ccrn4l) in mice, a gene that encodes a circadian deadenylase, confers resistance to diet-induced obesity. Mice lacking Nocturnin remain lean on high-fat diets, with lower body weight and reduced visceral fat. However, unlike lean lipodystrophic mouse models, these mice do not have fatty livers and do not exhibit increased activity or reduced food intake. Gene expression data suggest that Nocturnin knockout mice have deficits in lipid metabolism or uptake, in addition to changes in glucose and insulin sensitivity. Our data support a pivotal role for Nocturnin downstream of the circadian clockwork in the posttranscriptional regulation of genes necessary for nutrient uptake, metabolism, and storage.

Insulin Receptor Substrate 3 Is Not Essential for Growth or Glucose Homeostasis

The insulin receptor substrates (IRS) 1 and 2 are required for normal growth and glucose homeostasis in mice. To determine whether IRS-3, a recently cloned member of the IRS family, is also involved in the regulation of these, we have generated mice with a targeted disruption of the IRS-3 gene and characterized them. Compared with wild-type mice, the IRS-3-null mice showed normal body weight throughout development, normal blood glucose levels in the fed and fasted state and following an oral glucose bolus, and normal fed and fasted plasma insulin levels. IRS-3 is most abundant in adipocytes and is tyrosine-phosphorylated in response to insulin in these cells. Therefore, isolated adipocytes were analyzed for changes in insulin effects. Insulin-stimulated glucose transport in the adipocytes from the IRS-3-null mice was the same as in wild-type cells. The extent of tyrosine phosphorylation of IRS-1/2 following insulin stimulation was similar in adipocytes fromIRS-3-null and wild-type mice, and the insulin-induced association of tyrosine-phosphorylated IRS-1/2 with phosphatidylinositol 3-kinase and SHP-2 was not detectably increased by IRS-3 deficiency. Thus, IRS-3 was not essential for normal growth, glucose homeostasis, and glucose transport in adipocytes, and in its absence no significant compensatory augmentation of insulin signaling through IRS-1/2 was evident.

Muscle-specific deletion of rictor impairs insulin-stimulated glucose transport and enhances Basal glycogen synthase activity.

ABSTRACT Rictor is an essential component of mTOR (mammalian target of rapamycin) complex 2 (mTORC2), a kinase complex that phosphorylates Akt at Ser473 upon activation of phosphatidylinositol 3-kinase (PI-3 kinase). Since little is known about the role of either rictor or mTORC2 in PI-3 kinase-mediated physiological processes in adult animals, we generated muscle-specific rictor knockout mice. Muscle from male rictor knockout mice exhibited decreased insulin-stimulated glucose uptake, and the mice showed glucose intolerance. In muscle lacking rictor, the phosphorylation of Akt at Ser473 was reduced dramatically in response to insulin. Furthermore, insulin-stimulated phosphorylation of the Akt substrate AS160 at Thr642 was reduced in rictor knockout muscle, indicating a defect in insulin signaling to stimulate glucose transport. However, the phosphorylation of Akt at Thr308 was normal and sufficient to mediate the phosphorylation of glycogen synthase kinase 3 (GSK-3). Basal glycogen synthase activity in muscle lacking rictor was increased to that of insulin-stimulated controls. Consistent with this, we observed a decrease in basal levels of phosphorylated glycogen synthase at a GSK-3/protein phosphatase 1 (PP1)-regulated site in rictor knockout muscle. This change in glycogen synthase phosphorylation was associated with an increase in the catalytic activity of glycogen-associated PP1 but not increased GSK-3 inactivation. Thus, rictor in muscle tissue contributes to glucose homeostasis by positively regulating insulin-stimulated glucose uptake and negatively regulating basal glycogen synthase activity.

Characterization of the Insulin-regulated Membrane Aminopeptidase in 3T3-L1 Adipocytes

A novel membrane aminopeptidase has been identified as a major protein in vesicles from rat adipocytes containing the glucose transporter isotype Glut4. In this study we have characterized this aminopeptidase, referred to as vp165, in 3T3-L1 adipocytes. The subcellular distributions of vp165 and Glut4 were determined by immunoisolation of vesicles with antibodies against both proteins, by immunofluorescence, and by subcellular fractionation and immunoblotting. Relative amounts of vp165 at the cell surface in basal and insulin-treated cells were assayed by cell surface biotinylation. These experiments showed that vp165 and Glut4 were entirely colocalized and that vp165 increased markedly at the cell surface in response to insulin, in a way similar to Glut4. When intact cells were assayed with a novel, membrane-impermeant fluorogenic substrate for vp165, we found that insulin stimulated aminopeptidase activity at the cell surface. This observation provides direct evidence for the functional consequence of vp165 translocation.

Inhibition of GLUT4 Translocation by Tbc1d1, a Rab GTPase-activating Protein Abundant in Skeletal Muscle, Is Partially Relieved by AMP-activated Protein Kinase Activation

Insulin increases glucose transport by stimulating the trafficking of intracellular GLUT4 to the cell surface, a process known as GLUT4 translocation. A key protein in signaling this process is AS160, a Rab GTPase-activating protein (GAP) whose activity appears to be suppressed by Akt phosphorylation. Tbc1d1 is a Rab GAP with a sequence highly similar to that of AS160 and with the same Rab specificity as that of AS160. The role of Tbc1d1 in regulating GLUT4 trafficking has been unclear. Our previous study showed that overexpressed Tbc1d1 inhibited insulin-stimulated GLUT4 translocation in 3T3-L1 adipocytes, even though insulin caused phosphorylation on its single canonical Akt motif. In the present study, we show in 3T3-L1 adipocytes that Tbc1d1 is only 1/20 as abundant as AS160, that knockdown of Tbc1d1 has no effect on insulin-stimulated GLUT4 translocation, and that overexpressed Tbc1d1 also inhibits GLUT4 translocation elicited by activated Akt expression. These results indicate that endogenous Tbc1d1 does not participate in insulin-regulated GLUT4 translocation in adipocytes and suggest that the GAP activity of Tbc1d1 is not suppressed by Akt phosphorylation. In addition, we discovered that Tbc1d1 is much more highly expressed in skeletal muscle than fat and that the AMP-activated protein kinase (AMPK) activator 5′-aminoimidazole-4-carboxamide ribonucleoside partially reversed the inhibition of insulin-stimulated GLUT4 translocation by overexpressed Tbc1d1 in 3T3-L1 adipocytes. 5′-Aminoimidazole-4-carboxamide ribonucleoside activation of the kinase AMPK is known to cause GLUT4 translocation in muscle. The above findings strongly suggest that Tbc1d1 is a component in the signal transduction pathway leading to AMPK-stimulated GLUT4 translocation in muscle.

12/15-Lipoxygenase Products Induce Inflammation and Impair Insulin Signaling in 3T3-L1 Adipocytes

Inflammation and insulin resistance associated with visceral obesity are important risk factors for the development of type 2 diabetes, atherosclerosis, and the metabolic syndrome. The 12/15‐lipoxygenase (12/15‐LO) enzyme has been linked to inflammatory changes in blood vessels that precede the development of atherosclerosis. The expression and role of 12/15‐LO in adipocytes have not been evaluated. We found that 12/15‐LO mRNA was dramatically upregulated in white epididymal adipocytes of high‐fat fed mice. 12/15‐LO was poorly expressed in 3T3‐L1 fibroblasts and was upregulated during differentiation into adipocytes. Interestingly, the saturated fatty acid palmitate, a major component of high fat diets, augmented expression of 12/15‐LO in vitro. When 3T3‐L1 adipocytes were treated with the 12/15‐LO products, 12‐hydroxyeicosatetranoic acid (12(S)‐HETE) and 12‐hydroperoxyeicosatetraenoic acid (12(S)‐HPETE), expression of proinflammatory cytokine genes, including tumor necrosis factor‐α (TNF‐α), monocyte chemoattractant protein 1 (MCP‐1), interleukin 6 (IL‐6), and IL‐12p40, was upregulated whereas anti‐inflammatory adiponectin gene expression was downregulated. 12/15‐LO products also augmented c‐Jun N‐terminal kinase 1 (JNK‐1) phosphorylation, a known negative regulator of insulin signaling. Consistent with impaired insulin signaling, we found that insulin‐stimulated 3T3‐L1 adipocytes exhibited decreased IRS‐1(Tyr) phosphorylation, increased IRS‐1(Ser) phosphorylation, and impaired Akt phosphorylation when treated with 12/15‐LO product. Taken together, our data suggest that 12/15‐LO products create a proinflammatory state and impair insulin signaling in 3T3‐L1 adipocytes. Because 12/15‐LO expression is upregulated in visceral adipocytes by high‐fat feeding in vivo and also by addition of palmitic acid in vitro, we propose that 12/15‐LO plays a role in promoting inflammation and insulin resistance associated with obesity.