The insulin receptor exists as two isoforms, A and B, that result from alternative splicing of exon 11 in the primary transcript. We have shown previously that the alternative splicing is developmentally and hormonally regulated. Consequently, these studies were instigated to identify sequences within the primary RNA transcript that regulate the alternative splicing. Minigenes containing exons 10, 11, and 12 and the intervening introns were constructed and transfected into HepG2 cells, which contain both isoforms of the insulin receptor. The cells were able to splice the minigene transcript to give both A (؊ exon 11) and B-like (؉ exon 11) RNAs. A series of internal deletions within intron 10 were tested for their ability to give A and B RNAs. Intron 10 contained two sequences that modulated exon 11 inclusion; a 48-nucleotide purine-rich sequence at the 5 end of intron 10 that functions as a splicing enhancer and causes an increase in exon 11 inclusion, and a 43-nucleotide sequence at the 3 end of intron 10 upstream of the branch point sequence that favors skipping of exon 11. Increasing the length of the polypyrimidine tract at the 3 end of intron 10 caused exon 11 to be spliced constitutively, indicating that a weak splice site is required for alternative splicing. Finally, point mutations, insertions, and deletions within exon 11 itself were able to regulate inclusion of the exon both positively and negatively. The human insulin receptor (IR)1 is encoded by a single gene that is located on chromosome 19 and composed of 22 exons. The mature IR exists as two isoforms, designated A and B, which result from alternative splicing of the primary transcript (1-3). The A isoform lacks exon 11, is expressed ubiquitously, and is the only isoform in lymphocytes, brain, and spleen; the B isoform contains exon 11 and is expressed predominantly in liver, muscle, adipocytes, and kidney (4 -6). Exon 11 is composed of 36 nucleotides that encode a 12-amino acid segment (residues 717-728) of the carboxyl terminus of the ␣-subunit of IR. A number of investigators have suggested that the isoform ratio could be altered in non-insulin-dependent diabetes mellitus (7-10), but other studies have produced conflicting results (11-14). We have found that alterations in isoform ratio in skeletal muscle were associated with hyperinsulinemia rather than diabetes (15). Similar results have been found in the rhesus monkey (16). Along these lines, Sell and co-workers (17) have shown that alternative splicing of the IR gene is regulated by insulin in the Fao hepatoma cell line. Furthermore, we have shown that the alternative splicing is hormonally and developmentally regulated in both the HepG2 hepatoma and 3T3-L1 adipocyte cell lines (18). The changes in splicing were accompanied by increases in insulin sensitivity, as measured by a number of parameters (19). These data indicate that regulation of the alternative splicing of the IR is important for insulin sensitivity and responsiveness.Splicing of pre-mRNA depends on the presence of relatively...

Phosphatidylinositol 3-kinase (PI3K) activation is necessary for insulin-responsive glucose transporter (GLUT4) translocation and glucose transport. Insulin and platelet-derived growth factor (PDGF) stimulate PI3K activity in 3T3-L1 adipocytes, but only insulin is capable of stimulating GLUT4 translocation and glucose transport. We found that PDGF causes serine/threonine phosphorylation of insulin receptor substrate 1 (IRS-1) in 3T3-L1 cells, measured by altered mobility on SDSpolyacrylamide gel, and this leads to a decrease in insulin-stimulated tyrosine phosphorylation of IRS-1. The PI3K inhibitors wortmannin and LY294002 inhibit the PDGF-induced phosphorylation of IRS-1, whereas the MEK inhibitor PD98059 was without a major effect. PDGF pretreatment for 60 -90 min led to a marked 80 -90% reduction in insulin stimulatable phosphotyrosine and IRS-1-associated PI3K activity. We examined the functional consequences of this decrease in IRS-1-associated PI3K activity. Interestingly, insulin stimulation of GLUT4 translocation and glucose transport was unaffected by 60 -90 min of PDGF preincubation. Furthermore, insulin activation of Akt and p70 s6kinase , kinases downstream of PI3K, was unaffected by PDGF pretreatment. Wortmannin was capable of blocking these insulin actions following PDGF pretreatment, suggesting that PI3K was still necessary for these effects. In conclusion, 1) PDGF causes serine/threonine phosphorylation of IRS-1, and PI3K, or a kinase downstream of PI3K, mediates this phosphorylation. 2) This PDGF-induced phosphorylation of IRS-1 leads to a significant decrease in insulin-stimulated PI3K activity. 3) PDGF has no effect on insulin stimulation of Akt, p70 s6kinase , GLUT4 translocation, or glucose transport. 4) This suggests the existence of an IRS-1-independent pathway leading to the activation of PI3K, Akt, and p70 s6kinase ; GLUT4 translocation; and glucose transport.Tyrosine kinase receptors such as the insulin receptor, the epidermal growth factor (EGF) 1 receptor, and the plateletderived growth factor (PDGF) receptor activate many of the same signaling cascades. The two most prominent shared pathways are the phosphatidylinositol 3-kinase (PI3K) and the microtubule-associated protein kinase (MAPK) pathways. Activation of MAPK leads to mitogenic progression and may be involved in differentiation (for review, see Ref. 1). Numerous studies using inhibitors of PI3K activity, expression of constitutively active PI3K, and microinjection of dominant negative inhibitors of PI3K have all demonstrated that PI3K is necessary for the mitogenic effects of many growth factors and is both necessary and sufficient for the metabolic effects of insulin (2-7). PI3K is composed of two subunits, an 85-kDa regulatory subunit (p85) and a 110-kDa catalytic subunit (p110). The p85 subunit is composed of an N-terminal Src-homology 3 (SH3) domain and 2 Src-homology 2 (SH2) domains. The SH2 domains flank the region where the p110 associates with p85. The SH2 domains interact with phosphotyrosine residues, leading to sub...

Phospholipase C-␥ (PLC␥) is the isozyme of PLC phosphorylated by multiple tyrosine kinases including epidermal growth factor, platelet-derived growth factor, nerve growth factor receptors, and nonreceptor tyrosine kinases. In this paper, we present evidence for the association of the insulin receptor (IR) with PLC␥. Precipitation of the IR with glutathione S-transferase fusion proteins derived from PLC␥ and coimmunoprecipitation of the IR and PLC␥ were observed in 3T3-L1 adipocytes. To determine the functional significance of the interaction of PLC␥ and the IR, we used a specific inhibitor of PLC, U73122, or microinjection of SH2 domain glutathione S-transferase fusion proteins derived from PLC␥ to block insulin-stimulated GLUT4 translocation. We demonstrate inhibition of 2-deoxyglucose uptake in isolated primary rat adipocytes and 3T3-L1 adipocytes pretreated with U73122. Antilipolytic effect of insulin in 3T3-L1 adipocytes is unaffected by U73122. U73122 selectively inhibits mitogen-activated protein kinase, leaving the Akt and p70 S6 kinase pathways unperturbed. We conclude that PLC␥ is an active participant in metabolic and perhaps mitogenic signaling by the insulin receptor in 3T3-L1 adipocytes. The insulin receptor (IR)1 is a hetero-tetramer consisting of two ␣-subunits that are entirely extracellular and two ␤-subunits that span the plasma membrane and contain intrinsic tyrosine kinase activity (1, 2). One of the major metabolic effects of insulin in fat and skeletal muscle is the stimulation of glucose uptake (3). This occurs through the translocation of glucose transporters (GLUT4) from intracellular vesicles to the plasma membrane (4). Neither the molecular mechanism by which GLUT4 vesicles fuse with the plasma membrane nor the signaling proteins downstream of the IR leading to the stimulation of glucose transport have been clearly elucidated. An involvement of IRS-1 is indicated by both in vitro studies where primary rat adipocytes were transfected with an antisense ribozyme directed against rat IRS-1 (5) and in vivo studies where insulin-mediated glucose transport was attenuated in mice with targeted disruption of the IRS-1 gene (6). The ability of IRS-1 knock-out mice to transport glucose in response to insulin implies alternative mechanisms of glucose transport activation by insulin. PI 3-kinase has been demonstrated to be required for the insulin effect on glucose transport (7-10).Protein kinase C has been studied extensively as a mediator of insulin-stimulated glucose transport (11). The insulinomimetic effect of phorbol esters on glucose uptake implicates DAG as a potentiator of glucose uptake. Phorbol ester down-regulation reportedly inhibits insulin-stimulated glucose uptake in mouse soleus (12), rat heart (13), and rat adipocytes (14 -16). In 3T3-L1 adipocytes, however, insulin-stimulated glucose uptake has been reported to be refractory to down-regulation by phorbol esters (17,18). There are a number of ways that DAG can be generated in the cell in response to cell-surface receptors. An imme...

Interaction of the activated insulin receptor (IR) with its substrate, insulin receptor substrate 1 (IRS-1), via the phosphotyrosine binding domain of IRS-1 and the NPXY motif centered at phosphotyrosine 960 of the IR, is important for IRS-1 phosphorylation. We investigated the role of this interaction in the insulin signaling pathway that stimulates glucose transport. Utilizing microinjection of competitive inhibitory reagents in 3T3-Ll adipocytes, we have found that disruption of the IR/IRS-1 interaction has no effect upon translocation of the insulin-responsive glucose transporter (GLUT4). The activity of these reagents was demonstrated by their ability to block insulin stimulation of two distinct insulin bioeffects, membrane ruffling and mitogenesis, in 3T3-L1 adipocytes and insulin-responsive rat 1 fibroblasts. These data suggest that phosphorylated IRS-1 is not an essential component of the metabolic insulin signaling pathway that leads to GLUT4 translocation, yet it appears to be required for other insulin bioeffects.The regulation of plasma glucose levels is one of the major roles of insulin. An important aspect of this process is the stimulation of increased glucose transport into insulinresponsive tissues, which is primarily achieved by translocation of the insulin-responsive glucose transporter (GLUT4) from an intracellular pool to the plasma membrane. However, the intracellular signaling pathway between the insulin receptor (IR) and GLUT4 is not well defined. One signaling molecule predicted to play an essential role in this process is the major substrate of the IR tyrosine kinase, insulin receptor substrate 1 (IRS-1). The cDNA encoding IRS-1 has been isolated from several sources, revealing a coding sequence that is highly conserved across species (1-5) and distributed over a broad range of tissues. Insulin stimulation of most cell types leads to rapid phosphorylation of this 165,000-185,000 Mr protein on multiple tyrosine residues (6). Phosphorylation of IRS-1 occurs via interaction with an NPXY phosphotyrosine motif at position 960 of the autophosphorylated IR. The importance of this interaction was illustrated by studies of a mutant IR with a substitution of Phe for Tyr-960. The mutant receptor had normal kinase activity, yet did not significantly phosphorylate IRS-1 and had no biological activity when expressed in Chinese hamster ovary (CHO) cells (7). Further insight into the association between IRS-1 and the NPXY of the IR has been gained by studies using the yeast two-hybrid system. These studies demonstrated that amino acids 108-516 of IRS-1 are sufficient for binding phosphotyrosine 960 of the IR and are required for phosphorylation of IRS-1 by the IR (8-11). Furthermore, the recent cloning of IRS-2 has revealed extensive homology between IRS-1 and IRS-2 from amino acids 144-316, suggesting a minimal IR binding domain (12). This phosphotyrosine binding (PTB) domain is distinct from src homology 2 (SH2) domains. Although both IRS-1 and src homology and collagen have been shown to int...

The idea of ecotourism is being promoted and supported, by growing numbers of people and groups in different parts of the world, as a major means of dealing with the damaging effects of tourism. Yet the meaning of the term varies among different people, projects, and places. Evidence from national parks, where this type of tourism has been promoted for many years, shows that such tourism can cause substantial long-term cumulative changes in environment. Concepts such as ecotourism, green tourism, and sustainable tourism development, are general in their nature and have to be described, planned, and assessed, in detail on the ground in terms of the socioeconomic and environmental conditions applying in different places. In this respect, careful planning and management procedures are needed not only for ecotourism but indeed for all forms of tourism.