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.

Major histocompatibility complex (MHC) class I molecules present peptides, produced through cytosolic proteasomal degradation of cellular proteins, to cytotoxic T lymphocytes. In dendritic cells, the peptides can also be derived from internalized antigens through a process known as cross-presentation. The cellular compartments involved in cross-presentation remain poorly defined. We found a role for peptide trimming by insulin-regulated aminopeptidase (IRAP) in cross-presentation. In human dendritic cells, IRAP was localized to a Rab14+ endosomal storage compartment in which it interacted with MHC class I molecules. IRAP deficiency compromised cross-presentation in vitro and in vivo but did not affect endogenous presentation. We propose the existence of two pathways for proteasome-dependent cross-presentation in which final peptide trimming involves IRAP in endosomes and involves the related aminopeptidases in the endoplasmic reticulum.

OBJECTIVERictor 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 METHODSInsulin signaling and glucose and lipid metabolism were studied in FRic−/− fat cells. In vivo glucose metabolism was evaluated by hyperinsulinemic-euglycemic clamp.RESULTSLoss 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.CONCLUSIONSRictor/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.

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 circadiancontrolled 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 dietinduced 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.C ircadian clocks are present in most tissues of the body, where they control the expression of 5-10% of the tissue-specific mRNAs through both transcriptional and posttranscriptional regulation (1, 2). The widespread importance of circadian clock regulation is evident in that generalized disruption of normal clock function results in tumor formation, sleep disorders, and metabolic problems (reviewed in refs. 3 and 4). For example, mutations in the central clock genes Clock or Bmal1 result in metabolic changes found in obesity and the metabolic syndrome (5-8), and numerous genes involved in fatty acid, cholesterol, and glucose metabolism in liver are regulated in circadian or diurnal patterns (9-15), indicating that the clock plays a broad role in regulating metabolism. Nonetheless, the large number of genes, metabolic pathways, and cell/tissue types that are under general circadian control impose a major challenge in understanding the molecular details. Further advances in this area require refined understanding of the specific circadian output pathways by which the clocks regulate physiology.For cycling mRNAs to closely reflect daily rhythmic transcriptional drive, their half-lives must be relatively short. There are several examples of rhythmic posttranscriptional regulation in which the mRNA half-life or adenylation state changes over the course of the day (16-19), but very little is known about the mechanisms responsible. A likely contributor is Nocturnin (Ccrn4l, Noc), which has been implicated in the posttranscriptional regulation of mRNA stability and/or translatability by the circadian clock (20). Noc is expressed rhythmically in many tissues, with particularly high-amplitude rhythms in liver where mRNA levels are increased 100-fold in early night (21). Noc is at a pivotal position to play a role in shaping the rhythmic pattern of gene ...

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][191][192][193][194][195][196][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 C␥. 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.The insulin receptor is a tyrosine kinase, which when activated by insulin binding phosphorylates cellular substrates. The most well characterized of these are two members of the IRS 1 family, IRS-1 and IRS-2, and the protein Shc. Tyrosine phosphorylation of the IRS proteins creates binding sites for SH2 domain-containing signaling molecules, including PI 3-kinase, the adapter molecule Grb-2, and the protein-tyrosine phosphatase SHP-2. Docking of these proteins in turn activates specific signal transduction pathways (reviewed in Refs. 1 and 2). Recently, we have identified, by purification and cloning, a third member of the IRS family, called IRS-3, which in insulintreated adipocytes is tyrosine-phosphorylated and associated with PI 3-kinase (3, 4). All three IRS family members possess a common domain structure that includes PH and PTB domains at the N terminus and, C-terminal to these, a number of potential tyrosine phosphorylation sites (1,2,4,5). The presence of these features can therefore be viewed as defining an IRS. Previously, we have identified a 160-kDa protein in HEK 293 cells, termed PY160, which is rapidly tyrosine-phosphorylated in response to insulin but which is immunologically unrelated to IRS-1 (6). In the present study we have isolated PY160 from insulin-treated HEK 293 cells and cloned its cDNA. The predicted amino acid sequence shows that PY160 is a new member of the IRS family. EXPERIMENTAL PROCEDURESCell Culture and Preparation of Lysates-HEK 293 cells were grown on 10-cm plates as described previously (6). Before use, confluent plates of cells were incubated in serum-free medium for 2 h and then incubated for 5 min further with either no addition or the addition of 1 M insulin to activate fully the insulin and IGF-1 receptors present on these cells (7). Each plate was ...

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.

Tight control of glucose uptake in skeletal muscles and adipocytes is crucial to glucose homeostasis and is mediated by regulating glucose transporter GLUT4 subcellular distribution. In cultured cells, Rab GAP AS160 controls GLUT4 intracellular retention and release to the cell surface and consequently regulates glucose uptake into cells. To determine AS160 function in GLUT4 trafficking in primary skeletal muscles and adipocytes and investigate its role in glucose homeostasis, we characterized AS160 knockout (AS160(-/-)) mice. We observed increased and normal basal glucose uptake in isolated AS160(-/-) adipocytes and soleus, respectively, while insulin-stimulated glucose uptake was impaired and GLUT4 expression decreased in both. No such abnormalities were found in isolated AS160(-/-) extensor digitorum longus muscles. In plasma membranes isolated from AS160(-/-) adipose tissue and gastrocnemius/quadriceps, relative GLUT4 levels were increased under basal conditions and remained the same after insulin treatment. Concomitantly, relative levels of cell surface-exposed GLUT4, determined with a glucose transporter photoaffinity label, were increased in AS160(-/-) adipocytes and normal in AS160(-/-) soleus under basal conditions. Insulin augmented cell surface-exposed GLUT4 in both. These observations suggest that AS160 is essential for GLUT4 intracellular retention and regulation of glucose uptake in adipocytes and skeletal muscles in which it is normally expressed. In vivo studies revealed impaired insulin tolerance in the presence of normal (male) and impaired (female) glucose tolerance. Concurrently, insulin-elicited increases in glucose disposal were abolished in all AS160(-/-) skeletal muscles and liver but not in AS160(-/-) adipose tissues. This suggests AS160 as a target for differential manipulation of glucose homeostasis.

The nuclear receptor peroxisome proliferator-activated receptor ␥ (PPAR-␥) is an important target in diabetes therapy, but its direct role, if any, in the restoration of islet function has remained controversial. To identify potential molecular mechanisms of PPAR-␥ in the islet, we treated diabetic or glucose-intolerant mice with the PPAR-␥ agonist pioglitazone or with a control. Treated mice exhibited significantly improved glycemic control, corresponding to increased serum insulin and enhanced glucose-stimulated insulin release and Ca 2؉ responses from isolated islets in vitro. This improved islet function was at least partially attributed to significant upregulation of the islet genes Irs1, SERCA, Ins1/2, and Glut2 in treated animals. The restoration of the Ins1/2 and Glut2 genes corresponded to a two-to threefold increase in the euchromatin marker histone H3 dimethyl-Lys4 at their respective promoters and was coincident with increased nuclear occupancy of the islet methyltransferase Set7/9. Analysis of diabetic islets in vitro suggested that these effects resulting from the presence of the PPAR-␥ agonist may be secondary to improvements in endoplasmic reticulum stress. Consistent with this possibility, incubation of thapsigargin-treated INS-1 ␤ cells with the PPAR-␥ agonist resulted in the reduction of endoplasmic reticulum stress and restoration of Pdx1 protein levels and Set7/9 nuclear occupancy. We conclude that PPAR-␥ agonists exert a direct effect in diabetic islets to reduce endoplasmic reticulum stress and enhance Pdx1 levels, leading to favorable alterations of the islet gene chromatin architecture.Type 2 diabetes mellitus results from a combination of insulin resistance and progressive islet dysfunction (46). In many individuals, ␤-cell failure may precede the clinical diagnosis of diabetes, and landmark studies such as the United Kingdom Prospective Diabetes Study have shown a continued decrement in ␤-cell function despite treatment intervention with sulfonylureas, metformin, and insulin (52). Thiazolidinediones are orally active agents used in the treatment of type 2 diabetes that act as agonists for the nuclear transcription factor peroxisome proliferator-activated receptor ␥ (PPAR-␥) (60). Although thiazolidinediones are classically thought to act as peripheral insulin sensitizers, there is growing evidence from studies of human and animal models that these agents may also act to preserve and/or enhance ␤-cell function in the setting of progressive type 2 diabetes and insulin resistance (3, 12). PPAR-␥ is known to be expressed in the pancreatic islet (8, 48), and PPAR-responsive elements have been identified in the promoters of genes involved in glucose-stimulated insulin secretion, including Glut2, Gck, and Pdx1 (16,21,26,27,33). Reports from studies of ␤-cell lines, rodent models of progressive type 2 diabetes, and humans at risk for type 2 diabetes suggest that PPAR-␥ agonist administration leads to preservation of islet mass and function (10,13,18,22,25,33,57,58).Whereas the studies noted ab...