Biological action of leptin as an angiogenic factor.

Leptin is a hormone that regulates food intake, and its receptor (OB-Rb) is expressed primarily in the hypothalamus. Here, it is shown that OB-Rb is also expressed in human vasculature and in primary cultures of human endothelial cells. In vitro and in vivo assays revealed that leptin has angiogenic activity. In vivo, leptin induced neovascularization in corneas from normal rats but not in corneas from fa/fa Zucker rats, which lack functional leptin receptors. These observations indicate that the vascular endothelium is a target for leptin and suggest a physiological mechanism whereby leptin-induced angiogenesis may facilitate increased energy expenditure.

Identification of a novel hypothalamic neuropeptide Y receptor associated with feeding behavior.

Neuropeptide Y (NPY) plays important roles in the central control of appetite and energy balance, but the receptor subtype responsible for this function has not been cloned. Here we report the cloning by expression of a novel NPY receptor subtype from a rat hypothalamus cDNA library. The novel receptor, referred to as the NPY Y5 receptor, has a transcript of approximately 2.6 kilobases with an open reading frame of 1335 base pairs that encodes a 445-amino acid protein. The amino acid sequence deduced from the rat Y5 cDNA clone shows only 30-33% identity to other NPY receptors, including Y1, Y2, and Y4/PP1. Using the rat Y5 receptor cDNA probe, the human homologue was obtained by low stringency hybridization. The human Y5 amino acid sequence has 88% identity to the rat Y5 receptor. Importantly, pharmacological analysis shows that the rat and human Y5 receptors have high affinity for the peptides that elicit feeding (e.g. NPY, PYY, (2-36)NPY, and (LP)NPY) and low affinity for nonstimulating peptides (e.g. (13-36)NPY and rat PP), suggesting that it is the NPY feeding receptor subtype.

Cyclic AMP-induced transcriptional repression of the insulin-responsive glucose transporter (GLUT4) gene: identification of a promoter region required for down-regulation of transcription.

The mechanism(s) by which cyclic AMP represses transcription of the GLUT4 gene was investigated. 3T3-L1 preadipocytes were stably transfected with a series of 5' deletion mutants of the mouse GLUT4 gene promoter fused to the bacterial CAT gene and then were induced to differentiate into adipocytes. A method based on reverse transcription/polymerase chain reaction (PCR) amplification was developed and optimized to quantitate expression of CAT mRNA transcripts. Treatment with 8-bromo-cAMP down-regulated the level of CAT mRNA in adipocytes transfected with the -7000/CAT, -785/CAT and -469/CAT constructs, but not the -78/CAT construct. Thus, the regulatory element(s) which mediates transcriptional repression by cAMP resides in the proximal promoter of the GLUT4 gene between positions -469 and -78. Since down-regulation of GLUT4 mRNA is unaffected by inhibitors of protein synthesis, cAMP (and insulin) may activate phosphorylation or dephosphorylation of an existing transcription factor that interacts with the GLUT4 proximal promoter.

Regulated expression of an insulin-responsive glucose transporter (GLUT4) minigene in 3T3-L1 adipocytes and transgenic mice.

Preliminary studies showed that up to 7 kb of 5' flanking sequence of the insulin-responsive glucose transporter (GLUT4) gene are insufficient to mediate differentiation-induced reporter gene expression in mouse 3T3-L1 preadipocytes. To locate the regulatory element(s) responsible for this function, a minigene containing the entire GLUT4 gene with substantial 5' and 3' flanking sequence and a short segment of foreign DNA (for transcript identification) was constructed and transfected into mice and 3T3-L1 preadipocytes at relatively low copy number. In transgenic mice the GLUT4 minigene exhibited a pattern of tissue-specific expression similar, but not identical, to that of the endogenous gene. In 3T3-L1 cells expression of minigene mRNA occurred upon differentiation into adipocytes, with kinetics virtually identical to that of endogenous GLUT4 mRNA. In both cultured adipocytes and transgenic mice, the level of expression of the minigene was low relative to that of the endogenous gene. Treatment of minigene-transfected 3T3-L1 adipocytes with 8-bromo-cAMP, which represses transcription of the endogenous GLUT4 gene, also repressed expression of the GLUT4 minigene. However, insulin, which down-regulates transcription of the endogenous GLUT4 gene, failed to normally down-regulate expression of the GLUT4 minigene. These findings indicate that the cis-acting elements required for directing tissue-specific expression (in heart, skeletal muscle, and brown adipose tissue), differentiation-induced activation of transcription, and cAMP-induced repression of transcription are located within the 14-kb GLUT4 minigene. However, the cis elements necessary for maximal tissue-specific expression and for insulin-induced down-regulation of expression are not located in the minigene.

Insulin down-regulates expression of the insulin-responsive glucose transporter (GLUT4) gene: effects on transcription and mRNA turnover.

Insulin rapidly represses expression of the gene encoding the insulin-responsive glucose transporter (GLUT4) in 3T3-L1 mouse adipocytes. Upon exposure to the hormone the cellular level of GLUT4 mRNA falls (t1/2 approximately 2.5 hr) to 20-30% of its initial level within 10 hr. This is followed by a similar decrease in the level of GLUT4 protein. Down-regulation of GLUT4 mRNA is a result of both rapid repression of transcription of the GLUT4 gene and an increased rate of turnover of the GLUT4 message. As a consequence of prolonged exposure to insulin, 3T3-L1 adipocytes lose their capacity for acute stimulation of hexose uptake by insulin. These findings provide an explanation for the resistance of glucose uptake to insulin in adipose tissue observed in non-insulin-dependent (type 2) diabetes mellitus, particularly that associated with hyperinsulinemia and obesity.

The insulin-like growth factor 1 (IGF-1) receptor is responsible for mediating the effects of insulin, IGF-1, and IGF-2 in Xenopus laevis oocytes.

Competitive hormone binding studies with membrane and partially purified receptors from Xenopus laevis oocytes revealed that the oocyte possesses high affinity (KD = 1-3 nM) binding sites for both insulin growth factors 1 and 2 (IGF-1 and IGF-2), but not for insulin. Consistent with these findings, IGF-1 activates hexose uptake by Xenopus oocytes with a KA (3 nM) identical with its KD, while IGF-2 and insulin activate hexose uptake with KA values of 50 nM and 200-250 nM, respectively, suggesting activation mediated through an IGF-1 receptor. Both IGF-1 and insulin activate receptor beta-subunit autophosphorylation and, thereby, protein substrate (reduced and carboxyamidomethylated lysozyme, i.e. RCAM-lysozyme) phosphorylation with KA values comparable to their respective KD values for ligand binding and KA values for activation of hexose uptake. The autophosphorylated beta-subunit(s) of the receptor were resolved into two discrete components, beta 1 and beta 2 (108 kDa and 94 kDa, respectively), which were phosphorylated exclusively on tyrosine and which exhibited similar extents of IGF-1-activated autophosphorylation. When added prior to autophosphorylation, RCAM-lysozyme blocks IGF-1-activated autophosphorylation and, thereby, IGF-1-activated protein substrate (RCAM-lysozyme) phosphorylation. Based on these findings, we conclude that IGF-1-stimulated autophosphorylation of its receptor is a prerequisite for catalysis of protein substrate phosphorylation by the receptor's tyrosine-specific protein kinase. The IGF-1 receptor kinase is implicated in signal transmission from the receptor, since anti-tyrosine kinase domain antibody blocks IGF-1-stimulated kinase activity in vitro and, when microinjected into intact oocytes, prevents IGF-1-stimulated hexose uptake.

Transcriptional repression of the mouse insulin-responsive glucose transporter (GLUT4) gene by cAMP.

Glucose uptake by adipose tissue is mediated by two glucose transporters: GLUT4, which is most abundant, and GLUT1. While GLUT1 is expressed in many tissues, GLUT4 is unique to tissues that exhibit insulin-stimulated glucose uptake (heart and skeletal muscle and adipose tissue). In the diabetic state and during starvation, insulin-stimulated glucose uptake and GLUT4 expression are decreased in tissue adipocytes. Using 3T3-L1 adipocytes in culture, we investigated the possibility that these effects are mediated by elevated cellular cAMP. When 3T3-L1 adipocytes were treated for 16 hr with forskolin or 8-Br-cAMP, GLUT4 mRNA and protein were decreased by approximately 70%, while expression of GLUT1 mRNA and protein was increased 3-fold. These changes were accompanied by an increased basal rate of 2-deoxyglucose uptake and a loss of acute responsiveness of hexose uptake to insulin. The magnitude of GLUT4 mRNA depletion/GLUT1 mRNA accumulation was dependent upon the concentration of 8-Br-cAMP. The decrease of GLUT4 mRNA caused by 8-Br-cAMP was the result of a decreased transcription rate, while the half-life of the message was unaffected. The increase in GLUT1 mRNA caused by 8-Br-cAMP was the result of both transient transcriptional activation and mRNA stabilization. We suggest that down-regulation of GLUT4 mRNA in adipose tissue in the diabetic state and during starvation is the result of repression of transcription of the GLUT4 gene caused by cAMP.

Insulin receptor tyrosine kinase-catalyzed phosphorylation of 422(aP2) protein. Substrate activation by long-chain fatty acid.

It was established previously that the 15-kDa protein phosphorylated in 3T3-L1 adipocytes treated with insulin and phenylarsine oxide is O-phospho-Tyr19 422(aP2) protein, a fatty acid-binding protein. To assess its capacity to serve as substrate of the insulin receptor tyrosine kinase in vitro, native 422(aP2) protein was isolated from 3T3-L1 adipocytes and purified to homogeneity. Receptor-catalyzed phosphorylation of 422(aP2) protein on Tyr19 was markedly activated when long-chain fatty acid, e.g. oleic acid, is bound to the protein. Fatty acid had no effect on autophosphorylation of the insulin receptor by its intrinsic tyrosine kinase. Both saturated (C14, C16, and C18) and unsaturated (all cis-delta 9 C16, -delta 9 C18, and -delta 9,12 C18, -delta 9,12,15 C18, and -delta 5,8,11,14 C20) fatty acids caused substrate activation. The Km for 422(aP2) protein was greatly reduced (from 170 to 3 microM) by oleic acid with little or no effect on Vmax. Upon binding fatty acid to 422(aP2) protein the susceptibility of Tyr19 and Tyr128 to iodination by the lactoperoxidase method increased greatly. These results indicate that upon binding fatty acid, 422(aP2) protein undergoes a conformational change whereby Tyr19, which lies within a consensus-type sequence for tyrosine kinase substrates, becomes accessible for phosphorylation by the insulin receptor tyrosine kinase and to iodination by lactoperoxidase.

Insulin-receptor tyrosine kinase and glucose transport.

We identified the earliest events in autophosphorylation of the insulin receptor after insulin addition. Insulin-stimulated autophosphorylation at specific sites in the tyrosine kinase domain of the receptor's beta-subunit is correlated kinetically with activation of kinase-catalyzed phosphorylation of a model substrate (reduced and carboxyamidomethylated lysozyme; RCAM-lysozyme). To identify these sites, the deduced amino acid sequence of the 3T3-L1 adipocyte insulin receptor of the mouse was determined. Insulin-induced activation of substrate phosphorylation was shown to require autophosphorylation of three neighboring tyrosines (Tyr1148, Tyr1152, and Tyr1153) in the mouse receptor. A search for cellular substrates of the receptor kinase revealed that insulin causes accumulation of a 15,000-Mr phosphorylated (on tyrosine) cytosolic protein (pp15) in 3T3-L1 adipocytes treated with oxophenylarsine (PAO). PAO blocks turnover of the phosphoryl group of pp15, causing its accumulation, and thereby appears to interrupt signal transmission from the receptor to the glucose-transport system. Two membrane-bound protein phosphotyrosine phosphatases that are inhibited by PAO and are apparently responsible for the turnover of the pp15 phosphoryl group have been purified from 3T3-L1 adipocytes and characterized. These and other results support the hypothesis that turnover of the phosphoryl group of pp15, a product of insulin-receptor tyrosine kinase action, couples signal transmission to the glucose-transport system. [32P]pp15 was purified to homogeneity from 3T3-L1 adipocytes. Amino acid and radiochemical sequence analysis of the purified tryptic [32P]phosphopeptide revealed that pp15 is the phosphorylation product of 422(aP2) protein, a 15,000-Mr adipocyte protein whose cDNA we previously cloned and sequenced. 422(aP2) protein was found to bind fatty acids. When exposed to a free fatty acid, notably oleic acid, 422(aP2) protein becomes an excellent substrate of the isolated insulin-receptor tyrosine kinase. Compelling evidence indicates that on binding fatty acid, 422(aP2) protein undergoes a conformational change whereby Tyr19 becomes accessible to the receptor tyrosine kinase and undergoes O-phosphorylation. Adipose tissue and skeletal and heart muscle, which exhibit insulin-stimulated glucose uptake, express a specific insulin-responsive glucose transporter. A cDNA (GT2) that encodes this protein was isolated from a mouse 3T3-L1 adipocyte library and sequenced. We also isolated and characterized the corresponding mouse gene GLUT4. DNase I footprinting with nuclear extracts from 3T3-L1 cells revealed that a differentiation-specific nuclear factor binds to the GLUT4 promoter. The purified transcription factor C/EBP binds at the same position.(ABSTRACT TRUNCATED AT 400 WORDS)

Substrate phosphorylation catalyzed by the insulin receptor tyrosine kinase. Kinetic correlation to autophosphorylation of specific sites in the beta subunit.

The kinetics of insulin-stimulated autophosphorylation of specific tyrosines in the beta subunit of the mouse insulin receptor and activation of receptor kinase-catalyzed phosphorylation of a model substrate were compared. The deduced amino acid sequence of the mouse proreceptor was determined to locate tyrosine-containing tryptic peptides. Receptor was first incubated with unlabeled ATP to occupy nonrelevant autophosphorylation sites, after which [32P]autophosphorylation at relevant sites and attendant activation of substrate phosphorylation were initiated with [gamma-32P]ATP and insulin. Activation of substrate phosphorylation underwent an initial lag of 10-20 s during which there was substantial 32P-autophosphorylation of tryptic phosphopeptides p2 and p3, but not p1. Following the lag, incorporation of 32P into p1 and the activation of substrate phosphorylation increased abruptly and exhibited identical kinetics. The addition of substrate to the receptor prior to ATP inhibits insulin-stimulated autophosphorylation, and consequently substrate phosphorylation. Insulin-stimulated autophosphorylation of the receptor in the presence of substrate inhibited primarily the incorporation of 32P into p1 and drastically inhibited substrate phosphorylation. From Edman radiosequencing of 32P-labeled p1, p2, and p3 and the amino acid sequence of the mouse receptor, the location of each phosphopeptide within the beta subunit was determined. Further characterization of these phosphopeptides revealed that p1 and p2 represent the triply and doubly phosphorylated forms, respectively, of the region within the tyrosine kinase domain containing tyrosines 1148, 1152, and 1153. The doubly phosphorylated forms contain phosphotyrosines either at positions 1148 and 1152/1153 or positions 1152 and 1153. These results indicate that insulin stimulates sequential autophosphorylation of tyrosines 1148, 1152 and 1153, and that the transition from the doubly to the triply phosphorylated forms is primarily responsible for the activation of substrate phosphorylation.

Identification of phosphorylated 422(aP2) protein as pp15, the 15-kilodalton target of the insulin receptor tyrosine kinase in 3T3-L1 adipocytes.

[32P]pp15, the [32P]phosphorylated form of a specific cytosolic substrate of the insulin receptor tyrosine kinase, was purified to homogeneity from mouse 3T3-L1 adipocytes incubated with 32Pi. Evidence presented here and previously indicates that pp15 contains a single phosphotyrosine residue. Alkylated [32P]pp15 was subjected to limited digestion with trypsin, after which three incompletely digested tryptic [32P]phosphopeptides were purified for analysis. Amino acid and radiochemical sequence analysis of the [32P]phosphopeptides revealed that pp15 is the phosphorylation product of 422(aP2) protein, a 15-kDa adipocyte protein previously sequenced in this laboratory from the corresponding cDNA.