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SNARE proteins (VAMP2, syntaxin4, and SNAP23) have been thought to play a key role in GLUT4 trafficking by mediating the tethering, docking and subsequent fusion of GLUT4-containing vesicles with the plasma membrane. The precise functions of these proteins have remained elusive, however. We have now shown that depletion of the vesicle SNARE (v-SNARE) VAMP2 by RNA interference in 3T3-L1 adipocytes inhibited the fusion of GLUT4 vesicles with the plasma membrane but did not affect tethering of the vesicles to the membrane. In contrast, depletion of the target SNAREs (t-SNAREs) syntaxin4 or SNAP23 resulted in impairment of GLUT4 vesicle tethering to the plasma membrane. Our results indicate that the t-SNAREs syntaxin4 and SNAP23 are indispensable for the tethering of GLUT4 vesicles to the plasma membrane, whereas the v-SNARE VAMP2 is not required for this step but is essential for the subsequent fusion event.

In patients with various catabolic conditions, glucocorticoid excess induces skeletal muscle wasting by accelerating protein degradation via the ubiquitin-proteasome pathway. Although the transcriptional coactivator p300 has been implicated in this pathological process, regulatory mechanisms and molecular targets of its action remain unclear. Here we show that CREB-binding protein (CBP)/p300-interacting transactivator with ED-rich tail 2 (Cited2), which binds to the cysteine-histidine-rich region 1 of p300 and CBP, regulates muscle mass in vitro. Adenovirus-mediated overexpression of wild-type Cited2 significantly blocked morphological alterations of C2C12 myotubes with a concomitant decrease in myosin heavy chain protein in response to synthetic glucocorticoid dexamethasone, which were attributable to the reduced induction of atrophy-related ubiquitin ligases MuRF1 and MAFbx. These myotube-sparing effects were less pronounced, however, with a carboxyl-terminally truncated mutant of Cited2 that lacked the ability to bind p300. These results suggest that the gain of Cited2 function counteracts glucocorticoid-induced muscle atrophy through inhibition of proteolysis mediated by p300-dependent gene transcription.

KLF15 (Kruppel-like factor 15) plays a key role in adipocyte differentiation and glucose transport in adipocytes through activation of its target genes. We have now identified six target genes regulated directly by KLF15 in 3T3-L1 mouse adipocytes with the use of a combination of microarray-based chromatin immunoprecipitation and gene expression analyses. We confirmed the direct regulation by KLF15 of one of these genes, that for adrenomedullin, with the use of a luciferase reporter assay in 3T3-L1 preadipocytes and adipocytes. Such analysis revealed that the most proximal CACCC element in the promoter of the human adrenomedullin gene (located in the region spanning nucleotides −70 and −29) is required for trans-inhibition by KLF15. Furthermore, chromatin immunoprecipitation showed that KLF15 binds to this region of the human adrenomedullin gene promoter in cultured human adipocytes. These results thus implicate KLF15 in the regulation of adrenomedullin expression in adipose tissue.

The −112A > C polymorphism (rs10011540) of the gene for uncoupling protein 1 (UCP1) has been associated with type 2 diabetes mellitus in Japanese individuals. The aim of the present study was to investigate the effects of this polymorphism, as well as the well-known −3826A > G polymorphism (rs1800592), on clinical characteristics of type 2 diabetes. We determined the genotypes of the two polymorphisms in 93 Japanese patients with type 2 diabetes. Intramyocellular lipid content and hepatic lipid content (HLC) were measured by magnetic resonance spectroscopy. No significant differences in age, sex, BMI, or HbA1c level were detected between type 2 diabetic patients with the −112C allele and those without it. However, homeostasis model assessment for insulin resistance (p = 0.0089) and HLC (p = 0.012) was significantly greater in patients with the −112C allele. We did not detect an association of the −3826A > G polymorphism (rs1800592) of UCP1 gene with any measured parameters. These results suggest that insulin resistance caused by the −112C allele influences the susceptibility to type 2 diabetes.

Regulation of hepatic gene expression is important for energy homeostasis. We now show that hepatic expression of the gene for the transcription factor Kruppel-like factor 15 (KLF15) is increased by food deprivation and reduced by feeding in mice. Expression of the KLF15 gene in mouse liver was also down-regulated by a euglycemic–hyperinsulinemic clamp and was increased by inhibition of phosphatidylinositol 3-kinase. In cultured rat hepatocytes, KLF15 gene expression was induced by dexamethasone and a non-hydrolyzing analog of cAMP, and this effect was inhibited by insulin in a manner dependent on phosphatidylinositol 3-kinase signaling. Forced expression of KLF15 in cultured hepatocytes increased both the expression and the promoter activity of the gene for phosphoenolpyruvate carboxykinase (PEPCK). These results suggest that insulin and its counteracting hormones regulate the hepatic expression of KLF15, and that this transcription factor contributes to the regulation of PEPCK gene expression in the liver.

The mRNA expression of vascular endothelial growth factor (VEGF) was evaluated in colorectal adenocarcinomas and adjacent noncancerous colorectal tissues in 18 Japanese patients. The expression was confirmed to be up-regulated in the colorectal adenocarcinomas, when compared with the noncancerous tissues. Twelve genotypes of VEGF : six positions in the promoter region, two in the 5′UTR, and four in the 3′UTR, and their association with the expression of VEGF mRNA were evaluated. While G-1877A, T-1455C, G-1154A, C702T, and G1612A were not detected, C-2578A, T-1498C, G-1190A, C-634G, C-7T, C936T, and C1451T were found at allele frequencies of 4/36, 15/36, 15/36, 20/36, 8/36, 6/36, and 6/36, respectively, suggesting that C-2578A, G-1154A, and G1612A were associated with a decreased risk for colorectal adenocarcinoma. T-1498C (G-1190A) and C-7T were found to be associated with higher levels of VEGF mRNA, and may be a risk factor for the development of liver metastasis and/or prognosis of colorectal adenocarcinoma.

The formation of glucose-derived methylglyoxal (MG), a highly reactive dicarbonyl compound, is accelerated under diabetic conditions. We examined whether MG was capable of inducing apoptosis in Schwann cells (SCs), since recent studies have suggested a potential involvement of apoptotic cell death in the development of diabetic neuropathy. MG induced apoptosis in SCs in a dose-dependent manner, accompanied by a reduction of intracellular glutathione content and activation of the p38 MAPK. Inhibiting the p38 MAPK activation by SB203580 successfully suppressed the MG-induced apoptosis in SCs. Aminoguanidine and N-acetyl-L-cysteine also inhibited the MG-induced p38 MAPK activation and apoptosis along with restoration of the intracellular glutathione content. These results suggest a potential role for MG in SC injury through oxidative stress-mediated p38 MAPK activation under diabetic conditions, and it may serve as a novel insight into therapeutic strategies for diabetic neuropathy.

SNARE proteins have been implicated in the insulin-induced translocation of vesicles containing the GLUT4 glucose transporter to the plasma membrane of adipocytes. The role of the target SNARE SNAP-25 or its homologs in this process was investigated by screening a mouse adipocyte cDNA library with rat SNAP-25 and human SNAP-23 cDNA probes. Both positive clones isolated encoded a protein with 87% sequence identity to human SNAP-23, and which was therefore designated mouse SNAP-23. Immunoblot and immunofluorescence analyses revealed that SNAP-23 is located predominantly in the plasma membrane of 3T3-L1 adipocytes incubated in the absence or presence of insulin. Of syntaxins 1 to 5, SNAP-23 bound with the highest affinity to syntaxins 1 and 4 in the yeast two-hybrid system. Expression of SNAP-23, syntaxin 4, and the syntaxin-binding protein Munc 18c in COS cells revealed that Munc18c reduced the amount of SNAP-23 bound to syntaxin 4 in a concentration-dependent manner. These results suggest that the binding of SNAP-23 to syntaxin 4 is inhibited by Munc18c in adipocytes.

We investigated epidermal growth factor (EGF)-induced activation of 85-kDa/110-kDa phosphatidylinositol (PI)-3-kinase and 70-kDa S6 kinase in Chinese hamster ovary cells expressing the human EGF receptor. EGF-induced activation of p70 S6 kinase was comparable to that induced by insulin, whereas that of PI-3-kinase in anti-phosphotyrosine immunoprecipitates was very small compared with insulin. Wortmannin, a p85/p110 PI-3-kinase inhibitor, inhibited EGF-induced activation of p70 S6 kinase in a dose-dependent manner. Given that several proteins homologous to catalytic subunit of p85/p110 PI-3-kinase have been identified and that wortmannin inhibits distinct form of PI-3-kinase, the present results suggest that wortmannin-sensitive kinases that resemble catalytic subunit of p85/p110 PI-3-kinase may participate in the signaling pathway from EGF receptors to p70 S6 kinase.

Insulin stimulates glucose transport in muscle and fat cells by inducing the redistribution of a specific glucose transporter, GLUT4, from intracellular vesicles to the cell surface. Phosphoinositide (PI) 3-kinase has been implicated as a key intermediate in insulin-stimulated glucose transport by studies that have examined the effects of wortmannin and LY294002, which are thought to be specific inhibitors of this enzyme. However, the specificity of these compounds for PI 3-kinase has recently been questioned. Epidermal growth factor, which activates mitogen-activated protein kinase in mouse 3T3-L1 adipocytes, has now been shown to have no effect on PI 3-kinase activity or GLUT4 translocation in these cells. Furthermore, microinjection of a dominant negative mutant of the 85-kDa subunit of PI 3-kinase, which lacks a binding site for the catalytic 110-kDa subunit, inhibited GLUT4 translocation induced by insulin in 3T3-L1 adipocytes; microinjection of the wild-type protein had no effect. These observations indicate that PI 3-kinase is necessary for insulin-induced GLUT4 translocation and glucose transport in adipocytes.

The transcription factor sterol regulatory element-binding protein 1c (SREBP1c) plays an important role in the control of fatty acid metabolism in the liver. Evidence suggests that mammalian target of rapamycin (mTOR) complex 1 (mTORC1) contributes to the regulation of SREBP1c expression, but signaling downstream of mTORC1 remains unclear. We have now shown that medium rich in branched-chain amino acids stimulates expression of the SREBP1c gene in cultured hepatocytes in a manner sensitive both to rapamycin, a pharmacological inhibitor of mTORC1, and to a short hairpin RNA (shRNA) specific for S6 kinase 1 (S6K1), a downstream effector of mTORC1. The phosphorylation of S6K1 was increased in the liver of obese db/db mice. Furthermore, depletion of hepatic S6K1 in db/db mice with the use of an adenovirus vector encoding S6K1 shRNA resulted in down-regulation of SREBP1c gene expression in the liver as well as a reduced hepatic triglyceride content and serum triglyceride concentration. These results thus suggest that S6K1 regulates SREBP1c expression both in cultured hepatocytes and in mouse liver, and that increased hepatic activity of S6K1 contributes at least in part to the pathogenesis of obesity-induced hepatic steatosis and hypertriglyceridemia.

Summary To understand the molecular mechanism of gastric tumorigenesis, the status of neurofibromatosis type 1 (NF1) gene was analyzed in human gastric cancer cell lines. Although the sequencing of the GTPase activating protein (GAP)-related region of NF1 (NF1-GRD) revealed no apparent mutation, the NF1-GRD transcript (type I) and that containing an additional 63 bp insert in the center of NF1-GRD (type II) were equally expressed in most gastric cancer cells. By contrast, type II was predominantly expressed in normal stomach mucosa. When these two types of NF1-GRD were bacterially expressed and their GAP activities were tested, both types of NF1-GRD similarly stimulated ras GTPase activity. However, arachidonic acid inhibited GAP activities of two types of NF1-GRD to different extents. These results suggest that the increased expression of type I NF1 protein may modulate ras-related signal transduction and it may be related to the control of the gastric cellular proliferation.

Aim To study the changes in gene expression by pancreatic β cells under insulin resistance conditions. Method An exhaustive gene expression analysis was performed, using isolated pancreatic islets of obese diabetic model Lepr−/− mice. Overexpression of cyclin D2 was induced in cells from the pancreatic β cell line, namely, INS-1. Results Through a gene expression analysis using islets isolated from db/db mice, we found a significant increase in the expression of ribosome-related molecules. In addition, increased expression of cyclin D2 was found at certain protein levels. As INS-1 cells were induced to overexpress cyclin D2, we found an increase in the expression of ribosome-related molecules. Concurrently, an increase in the expression of endoplasmic reticulum stress (ER stress)-related molecules was also found. Conclusion In cases of pancreatic β cell hyperplasia associated with insulin resistance, ribosomal biogenesis is increased, and ER stress is induced.

The transcriptional regulator peroxisome proliferator-activated receptor-gamma coactivator-1alpha (PGC-1alpha) controls mitochondrial biogenesis and energy homeostasis. Although physical exercise induces PGC-1alpha expression in muscle, the underlying mechanism of this effect has remained incompletely understood. We recently identified a novel muscle-enriched isoform of PGC-1alpha transcript (designated PGC-1alpha-b) that is derived from a previously unidentified first exon. We have now cloned and characterized the human PGC-1alpha-b promoter. The muscle-specific transcription factors MyoD and MRF4 transactivated this promoter through interaction with a proximal E-box motif. Furthermore, either forced expression of Ca(2+)- and calmodulin-dependent protein kinase IV (CaMKIV), calcineurin A, or the p38 mitogen-activated protein kinase (p38 MAPK) kinase MKK6 or the intracellular accumulation of cAMP activated the PGC-1alpha-b promoter in cultured myoblasts through recruitment of cAMP response element (CRE)-binding protein (CREB) to a putative CRE located downstream of the E-box. Our results thus reveal a potential molecular basis for isoform-specific regulation of PGC-1alpha expression in contracting muscle.

The plasma enzyme, human lecithin-cholesterol acyltransferase(LCAT) is responsible for the majority of cholesterol ester formation in human plasma and is a key enzyme of the reverse transport of cholesterol from peripheral tissue to the liver. We sequenced genomic DNA of the LCAT gene from a Japanese male patient who was clinically and biochemically diagnosed as a familial LCAT deficiency. Analysis of all exons and exon-intron boundaries revealed only a single G to A transition within the sixth exon of both allele of the gene, leading to the substitution of methionine for isoleucinle at residue 293 of the mature enzyme. This mutation creates a new hexanucleotide recognition site for the restriction endonuclease Ndel. Familial study of Ndel digestion of the genomic DNA and determination of plasma LCAT activity established that the patient and his sister whose plasma LCAT activity were extremely reduced were homozygous and his children whose plasma LCAT activity were about half of normal controls were heterozygous for this mutation.

Skp2, the substrate-binding subunit of an SCF ubiquitin ligase complex, is a key regulator of cell cycle progression that targets substrates for degradation by the 26S proteasome. We have now shown that ablation of Skp2 in primary mouse embryonic fibroblasts (MEFs) results both in impairment of adipocyte differentiation and in the accumulation of the cyclin-dependent kinase inhibitor p27(Kip1), a principal target of the SCF(Skp2) complex. Genetic ablation of p27(Kip1) in MEFs promoted both lipid accumulation and adipocyte-specific gene expression. However, depletion of p27(Kip1) by adenovirus-mediated RNA interference failed to correct the impairment of adipocyte differentiation in Skp2(-/-) MEFs. In contrast, troglitazone, a high-affinity ligand for peroxisome proliferator-activated receptor gamma (PPARgamma), largely restored lipid accumulation and PPARgamma gene expression in Skp2(-/-) MEFs. Our data suggest that Skp2 plays an essential role in adipogenesis in MEFs in a manner that is at least in part independent of regulation of p27(Kip1) expression.

Insulin-degrading enzyme (IDE) accounts for most of the insulin degrading activity in extracts of several tissues and plays an important role in the intracellular degradation of insulin. Using newly developed sandwich radioimmunoassay for rat IDE, this enzyme was detectable in all tissues we examined and liver had the highest level of IDE. The ratio of insulin degrading activity to IDE concentration was roughly the same in liver, brain and muscle, however, twice as high in kidney as compared with other tissues. On the contrary, its degrading activity in these tissue extracts, including kidney, was completely lost after immunoprecipitation of IDE. These results suggest that IDE degrades insulin in the initial step of cleavage and that there are some mechanisms to regulate insulin degrading activity by IDE in the tissues.

Adiponectin is an anti-diabetic and anti-atherogenic hormone that is exclusively secreted from fat cells. Serum adiponectin levels are reduced in obese patients and obese model mice, despite increased adipose tissue mass. Elucidation of the mechanism(s) by which plasma adiponectin levels are decreased in obese and diabetic patients would provide insight into the cause of obesity-induced diabetes and the development of therapeutic advances. In the present study, the regulation of adiponectin secretion was investigated using 3T3-L1 adipocytes and a diabetic-/obese-mouse model. A novel insulin sensitizer, IkappaB kinase beta (IKKbeta) inhibitor, ameliorated insulin resistance and up-regulated plasma levels of adiponectin without producing a significant change in body weight in KKAy mice that were fed a high-fat diet. The IKKbeta inhibitor cancelled the TNFalpha-mediated down-regulation of adiponectin secretion and simultaneously up-regulated the phosphorylation of Akt in 3T3-L1 adipocytes. Using dominant-negative mutants of Akt or PKClambda (downstream effectors of phosphoinositide 3-kinase), insulin-stimulated Akt activity was found to be important in the regulation of adiponectin secretion by insulin in 3T3-L1 adipocytes. These observations suggest that "insulin-stimulated Akt activity in adipocytes" may play an important role in the regulation of adiponectin secretion.

In order to clarify the physiological relevance of the interaction between Shc and adaptins, components of plasma membrane-coated pit adaptor complex AP2, we investigated the role of Shc in ligand-induced endocytosis of epidermal growth factor (EGF) receptors. In vitro peptide binding assay showed that α-adaptin bound to the wild-type peptide corresponding to amino acids 346–355 of Shc, RDLFDMKPFE, but not to the mutant peptide in which both phenylalanines at 349 and 354 were substituted for alanines (FA). Using adenovirus vectors carrying a herpes simplex virus epitope-tagged 52-kDa wild-type Shc and Shc FA, we examined the interaction between Shc, AP2, and EGF receptors in intact cells. Alpha-adaptin bound to wild-type Shc in an EGF-dependent manner, whereas EGF-dependent association of α-adaptin with Shc FA was markedly reduced. In addition, EGF increased the amount of α-adaptin coprecipitated with EGF receptors in cells expressing wild-type Shc but not Shc FA. These results suggest that EGF stimulates Shc-AP2 complex formation and association of Shc-AP2 complexes with EGF receptors. Internalization assay showed that 125 I-EGF internalization was reduced in cells overexpressing Shc FA. Immunofluorescence study showed that punctate staining along the plasma membrane border as well as punctate pattern characteristic of cytoplasmic vesicles near the plasma membrane was enhanced in cells expressing wild-type Shc. These results suggest, therefore, the implication of Shc in ligand-induced endocytosis of EGF receptors in intact cells.

Peroxisome proliferator-activated receptor gamma (PPARgamma) is a nuclear receptor that is thought to be an important regulator of adipocyte differentiation. This ligand-dependent transcription factor is also activated by thiazolidinediones, a new class of synthetic antidiabetic drugs, resulting in a marked adipogenic response in cultured cells and enhanced insulin sensitivity in vivo. The importance of the COOH-terminal region of PPARgamma2 in thiazolidinedione-induced adipogenesis has now been investigated by expression of a mutant protein (PPARgamma2-DeltaC) that lacks the COOH-terminal 16 amino acids of full-length PPARgamma2. The mutant protein failed to bind a thiazolidinedione ligand, but its ability to bind the peroxisome proliferator response element was similar to that of the wild-type protein. Expression of PPARgamma2-DeltaC inhibited the thiazolidinedione-induced increase in trans-activation activity of endogenous PPARgamma in CV-1 cells. Furthermore, the mutant protein prevented thiazolidinedione-induced adipogenesis in 3T3-L1 cells, whereas expression of recombinant wild-type PPARgamma2 promoted adipogenesis. These data show not only that the COOH-terminal region of PPARgamma2 is indispensable for thiazolidinedione-induced adipogenesis mediated by this protein in 3T3-L1 cells, but also that the PPARgamma2-DeltaC mutant acts in a dominant negative manner by interfering with the access of endogenous PPARgamma to the peroxisome proliferator response element of target genes.

p70 S6 kinase plays an important role in growth factor-induced translational control and in cell cycle progression. Although the mechanism of p70 S6 kinase regulation is not fully understood, phosphorylation of serine and threonine residues of the enzyme is essential for its activation. The possible role of the serine-threonine kinase Akt in the activation of p70 S6 kinase induced by exposure of cells to heat has now been investigated. Overexpression of a mutant Akt1 (Akt-AA) in which the phosphorylation sites (Thr 308 and Ser 473 ) targeted by growth factors are replaced by alanine was shown to exert a dominant negative effect on Akt activation induced by platelet-derived growth factor (PDGF) or by heat treatment in CHO cells. Akt-AA also inhibited p70 S6 kinase activation induced by these stimuli. However, Akt-AA had no effect on the activation of p70 S6 kinase induced by 12- O -tetradecanoylphorbol 13-acetate, which did not stimulate Akt activity in these cells. These data suggest that Akt is required for heat treatment-induced activation of p70 S6 kinase.

SHPS-1 is a 120 kDa glycosylated receptor-like protein that contains immunoglobulin-like domains in its extracellular region and four potential tyrosine phosphorylation for SH2 domain binding sites in its cytoplasmic region. Epidermal growth factor (EGF) stimulated the rapid tyrosine phosphorylation of SHPS-1 and subsequent association of SHPS-1 with SHP-2, a protein tyrosine phosphatase containing SH2 domains, in Chinese hamster ovary cells overexpressing human EGF receptors. In the cells overexpressing SHPS-1, the tyrosine phosphorylation of SHPS-1 was more evident than that observed in parent cells. However, overexpression of SHPS-1 alone did not affect the activation of MAP kinase in response to EGF. These results suggest that SHPS-1 may be involved in the recruitment of SHP-2 from the cytosol to the plasma membrane in response to EGF.

SAP-1 is a human transmembrane-type protein tyrosine phosphatase that is abundant in colon and pancreatic cancer cell lines. The expression of SAP-1 in surgically excised human colorectal cancer specimens has now been investigated by immunohistochemical staining and in situ hybridization. Normal colon tissue or adenomas with mild dysplasia showed no detectable expression of SAP-1. In contrast, 2 of 17 adenomas with moderate or severe dysplasia and 19 of 48 (40%) adenocarcinomas expressed SAP-1, SAP-1 was localized predominantly to the apical surface of colonic cells in adenoma tissue showing moderate dysplasia, but it was also observed in the cytoplasm and at the basal surface of both adenoma cells showing severe dysplasia and adenocarcinoma cells. In situ hybridization with a SAP-1 antisense RNA probe detected SAP-1 mRNA in 6 of 9 colorectal cancers, but not in the surrounding mesenchymal tissue or normal mucosa. Sequencing of the K-RAS gene revealed that 10 of 15 (67%) SAP-1-positive cancers contained a mutation in codon 12. The serum concentration of carcinoembryonic antigen was normal in approximately half of the patients with SAP-1-positive colorectal cancers. These results suggest that SAP-1 is frequently overexpressed in human colorectal cancers and that such overexpression may occur relatively late in the adenoma-carcinoma sequence.

SHPS-1 (SHP substrate-1) is a glycosylated receptor-like protein with three immunoglobulin-like domains in its extracellular region and four YXX(L/V/I) motifs, potential tyrosine phosphorylation and SRC homology 2 (SH2) domain binding sites, in its cytoplasmic region. Various mitogens and cell adhesion induce tyrosine phosphorylation of SHPS-1 and its subsequent association with SHP-2, and SH2 domain-containing protein tyrosine phosphatase, suggesting that SHPS-1 plays a role in cell signaling in response to both growth factors and cell adhesion. The mouse and human cDNAs encoding SHPS-1 have now been isolated. The deduced amino acid sequences of rat, human, and mouse SHPS-1 show identities of 65 to 81%. In addition to the SH2 domain binding sites, a proline-rich putative SH3 domain binding site was detected in the cytoplasmic region of SHPS-1. Northern blot analysis revealed that human SHPS-1 mRNA is most abundant in brain and that the mouse mRNA is present in embryos as early as day 7. Fluorescence in situ hybridization localized the SHPS-1 gene to human chromosome 20p13 and the F3 band of mouse chromosome 2. Furthermore, interspecific backcross analysis placed the mouse SHPS-1 locus 5.0 centimorgans distal and 1.4 centimorgans proximal to the microsatellite markers D2Mit63 and D2Mit19, respectively, in a region associated with the mutations coloboma (Cm), lethal milk (lm), and well-haarig (we).

We have identified VAMP isoforms, VAMP-2 and cellubrevin, on GLUT4-containing vesicle membranes isolated from 3T3-L1 adipocytes. These proteins translocate from a low density microsomal fraction to the plasma membrane upon insulin stimulation in a fashion similar to GLUT4. VAMP-1 was not detected in this low density microsomal fraction nor on purified GLUT4-containing vesicles. In streptolysin-O permeabilized 3T3-L1 adipocytes, both VAMP-2 and cellubrevin were cleaved with botulinum neurotoxin isoform B, BoNTx/B. In addition, BoNTx/B partially inhibited insulin-stimulated GLUT4 translocation and glucose transport activity. We conclude that the synaptobrevin isoforms are important components of the insulin-dependent translocation of GLUT4 to the cell surface in adipocytes.

Binding proteins to the Src homology 3 (SH3) domains of Nck were screened by the use of glutathione S-transferase fusion proteins. Two proteins of 140 and 125 kDa were detected, both of which associated preferentially with the first SH3 domain of Nck. The 125-kDa protein, designated as Nap1 for Nck-associated protein 1, was purified and the corresponding rat cDNA was isolated. The predicted amino acid sequence revealed that p125 Nap1 dose not contain any known functional motif but shows sequence homology to Hem family gene. Using specific antibodies, p125 Nap1 was shown to associate with Nck both in vitro and in intact cells. Further characterization of p125 Nap1 may clarify the protein-protein interaction in the downstream signaling of Nck.

SH-PTP2 is a protein-tyrosine phosphatase with src homology-2 (SH2) domains and shown to be highly expressed in the rat brain. Light microscopic immunohistochemistry showed that specific immunoreactivity was widely distributed, most abundant in neurophil, weak in neuronal somata, and absent from white matter. By electron microscopic immunohistochemistry, intense labeling is observed on synapses and concentrated in the pre-and post-synaptic plasma membranes. In subcellular fractionation analysis of brain, SH-PTP2 was mainly observed in the particulate fraction, particularly in myelin and synaptosomes. SH-PTP2 was further recovered in the synaptic plasma membrane. SH-PTP2 was extracted from brain membrane with a detergent such as Triton X-100 or Nonidet P-40 but not with 1 M NaCl. Furthermore, SH-PTP2 was coimmunoprecipitated with a 100 kDa tyrosine-phosphorylated membrane protein, which may couple SH-PTP2 to brain membranes. These results suggest that SH-PTP2 associates with synaptic membranes and may play a role in the synaptic communications in the brain.

Summary To investigate the molecular pathogenesis of human gastric cancers the p53 gene, a suppressor oncogene, was analyzed in 12 human gastric cell lines. Southern blot and Northern blot analysis revealed a total deletion of p53 gene in KATO-III cells but no major abnormality of p53 gene in other cell lines. By the use of the reverse-transcriptase polymerase chain reaction and direct sequencing 7 cell lines showed point mutations of p53 gene resulting in amino-acid substitutions. Most of them were rare mutations which had not been observed in other types of cancers. One of these mutations was also detected through the use of PCR and oligomer-specific hybridization. Six out of 7 cell lines with mutations of p53 gene also lost one allele of chromosome 17p. Immunoblotting of cell lysates with an antibody specific to p53 demonstrated the absence of p53 protein in KATO-III cell. By contrast, the high levels of the p53 protein were observed in 5 cell lines all of which contained mutations of p53 gene. These results further suggest that the inactivation of p53 gene may play an important role in the transformation of gastric cells to the malignant phenotype. KATO-III cells might be a good model for studying the significance of the loss of p53 gene in cellular transformation.

The binding of cholecystokinin (CCK) to its receptors on guinea pig gastric chief cell membranes were characterized by the use of 125I-CCK-octapeptide (CCK8). At 30 degrees C optimal binding was obtained at acidic pH in the presence of Mg2+, while Na+ reduced the binding. In contrast to reports on pancreatic and brain CCK receptors, scatchard analysis of CCK binding to chief cell membranes revealed two classes of binding sites. Whereas, in the presence of a non-hydrolyzable GTP analog, GTP gamma S, only a low affinity site of CCK binding was observed. Chief cell receptors recognized CCK analogs, with an order of potency of: CCK8 greater than gastrin-I greater than CCK4. Although all CCK receptor antagonists tested (dibutyryl cyclic GMP, L-364718 and CR1409) inhibited labeled CCK binding to chief cell membranes, the relative potencies of these antagonists in terms of inhibiting labeled CCK binding were different from those observed in either pancreatic membranes or brain membranes. The results indicate, therefore, that on gastric chief cell membranes there exist specific CCK receptors, which are coupled to G protein. Furthermore, chief cell CCK receptors may be distinct from pancreatic or brain type CCK receptors.

Insulin gene expression is restricted to pancreatic B cells and the 5′ flanking region is responsible for the tissue specificity. The GCCATCTG motif in this region of the rat insulin 1 gene functions as an enhancer for insulin transcription. A cDNA coding for a GCCATCTG motif-binding protein (IEBP1) was isolated from a rat pancreatic B cell tumor γgt11 library. The IEBP1 protein was found to be the rat counterpart of the immunoglobulin (Ig) enhancer binding protein E12/47 having a helix-loop-helix domain. This result indicates that the Ig gene and insulin gene employ the same (or a similar) binding protein as a part of their transcriptional apparatus.

Erythropoietin (EPO) has a central role in the growth and development of erythroid cells. Using a biologically active radioiodinated derivative, EPO receptors were identified on fetal mouse liver cells mostly consisting of erythroid cells. 125 I-EPO was cross-linked to two receptor forms with apparent molecular masses of 110 and 95 kilodaltons, respectively and both having similar affinity toward EPO.

Summary In human epidermoid carcinoma KB cells, a glycoprotein of Mr=190,000 (gp190) has been shown to be phosphorylated on tyrosine residues upon EGF stimulation (Kadowaki et al., 1987, J. Biol. Chem. in press). Using a specific antibody to the c-terminal portion of the human c- erb B-2 gene product, we have found that gp190 is the human c- erb B-2 gene product which is structurally closely related to the epidermal growth factor (EGF) receptor. Since monoclonal antibody specific for the EGF receptor abolished both EGF binding to its receptor and tyrosine phosphorylation of the c-erbB-2 gene product, we have concluded that activation of EGF receptor tyrosine kinase activity upon EGF binding leads to the phosphorylation of the c- erb B-2 gene product on its tyrosine residues.

Insulin-induced tyrosine-phosphorylation in intact isolated rat adipocytes was studied using immunoblotting method with antiphosphotyrosine antibodies. Insulin-stimulated adipocytes were solubilized with Triton X-100. The lysate was incubated with wheat germ agglutinin, then with hydroxylapatite. Insulin stimulated tyrosine-phosphorylation of a 95 KDa protein which adsorbs to wheat germ agglutinin and appears to be the beta-subunit of the insulin receptor. Among the proteins adsorbed to hydroxylapatite, tyrosine-phosphorylation of 170 KDa and 60 KDa proteins was stimulated. 170 KDa was also stimulated by polyclonal anti-insulin receptor antibodies B-10 Ig G, IGF-I and H2O2. The detection of these proteins in rat adipocytes may lead to the elucidation of a common signal transduction pathway in insulin-responsive cells.

The effect of insulin on the phosphorylation of the insulin receptor was studied using isolated rat adipocytes. Rat adipocytes were labeled with [ 32 p] orthophosphate, treated with insulin for 15 min and solubilized in 1% Triton. The insulin receptor was then enriched by affinity chromatography on wheat germ lectin and identified by immunoprecipitation and NaDodSO 4 /acrylamide gel electrophoresis. Insulin (10 −8 M, 10 −7 M) stimulated the phosphorylation of the 95K subunit of the receptor 2 to 5-fold. This effect of insulin could also be shown in a cell-free system, i.e., if insulin was added after solubilization and wheat germ purification. In this cell-free system, insulin (3×10 −10 M to 10 −7 M) increased the incorporation of [ 32 p] from [γ- 32 p]-ATP into the 95K subunit of the receptor up to 16-fold and caused also a minor phosphorylation of the 135K subunit. By contrast, cyclic AMP (5×10 −6 M) and dibutyryl-cyclic AMP (10 −4 M) had no effect on basal or insulin stimulated receptor phosphorylation in the broken cell. These data suggest that insulin stimulation of phosphorylation of the insulin receptor is an early step in insulin action in adipocytes and is controlled by a non-cyclic AMP-dependent kinase.

A full-length cDNA clone that codes for glucose transporter protein was isolated from a rabbit brain cDNA library by using synthetic oligonucleotide probe derived from the sequence of human glucose transporter cDNA. The coding region shared 93.2% nucleotide and 97.0% amino-acid similarities with those of human glucose transporter and 89.4% nucleotide and 97.4% amino-acid similarities with those of rat transporter. Northern blot analysis revealed that glucose transporter mRNA is most abundant in the placenta and that it is also abundant in the brain. The fat tissue, heart, liver, and skeletal muscle of adult rats contained a very small amount of mRNA, while heart, liver, skeletal muscle and kidney of fetal rats contained a very high amount of glucose transporter mRNA. These results suggest that this type of glucose transporter might be closely related with cell proliferation and tissue development.

The band 3 glycoprotein from human erythrocytes was found to be phosphorylated on tyrosine residues by the purified EGF receptor kinase and the purified src kinase in vitro. Kinetic analysis revealed that Km of the band 3 protein phosphorylation by the EGF receptor kinase was 0.17 microM and 0.65 microM in the absence and presence of EGF (3 X 10(-7)M), respectively, and that in the case of the src kinase it was 0.4 microM. From these data the band 3 protein can be regarded as one of the best substrates common for the EGF receptor kinase and the src kinase in vitro.

A human myeloid leukemia cell line (HL-60) produces a peptide or peptides with insulin-like activity which is distinct from insulin or insulin-like growth factors (somatomedins). Factors regulating the production of this peptide (HL-ILP) were explored in the present study. The production of HL-ILP was maximal in the early log phase of cell growth and declined with increasing cell density. Differentiation of HL-60 cells to macrophages, induced by dihydroxyvitamin D 3 or phorbol esters, was also associated with a decrease in HL-ILP production. Glucose consumption by the cells in the early log phase was closely related with HL-ILP production, and HL-ILP was found to stimulate glucose consumption by HL-60 cells. Production of HL-ILP was dependent on glucose concentrations in the culture medium and glucose concentrations higher than 1mg/dl suppressed the release of HL-ILP. These observations are not inconsistent with a hypothesis that HL-ILP is involved in the glucose metabolism of the HL-60 cells that produce this peptide.

Antiserum to a defined region (residues 373-383) of the erbB oncogene product immunoprecipitated a 170,000 dalton protein that was phosphorylated in an EGF-sensitive fashion as well as the 125I-EGF-receptor complex from A431 human epidermoid carcinoma cells. Preincubation of the antiserum with an excess of the synthetic peptide corresponding to the defined region blocked the immunoprecipitation of this protein. A partial proteolytic peptide map of this immunoprecipitated 170,000 dalton protein was identical to that of the authentic EGF receptor. These results suggest immunological similarity between the erbB gene product and the EGF receptor.

Summary Fodrin (nonerythroid spectrin) from porcine brain was found to be phosphorylated on tyrosine residues by the purified insulin receptor kinase. The phosphorylation occurred in an insulin-sensitive manner with a physiologically relevant Km. The β (235 K) subunit of fodrin, but not the α (240 K) subunit, was phosphorylated by the kinase. Neither the α (240 K) subunit nor the β (220 K) subunit of erythrocyte spectrin was phosphorylated under the same conditions. Fodrin phosphorylation by the purified insulin receptor kinase was markedly inhibited by F-actin. These data raise the possibility that tyrosine phosphorylation of fodrin plays some roles in the regulation of plasma membrane-microfilament interaction.