Cortactin, an actin binding protein, regulates GLUT4 translocation via actin filament remodeling.

Insulin regulates glucose uptake into fat and skeletal muscle cells by modulating the translocation of GLUT4 between the cell surface and interior. We investigated a role for cortactin, a cortical actin binding protein, in the actin filament organization and translocation of GLUT4 in Chinese hamster ovary (CHO-GLUT4myc) and L6-GLUT4myc myotube cells. Overexpression of wild-type cortactin enhanced insulin-stimulated GLUT4myc translocation but did not alter actin fiber formation. Conversely, cortactin mutants lacking the Src homology 3 (SH3) domain inhibited insulin-stimulated formation of actin stress fibers and GLUT4 translocation similar to the actin depolymerizing agent cytochalasin D. Wortmannin, genistein, and a PP1 analog completely blocked insulin-induced Akt phosphorylation, formation of actin stress fibers, and GLUT4 translocation indicating the involvement of both PI3-K/Akt and the Src family of kinases. The effect of these inhibitors was even more pronounced in the presence of overexpressed cortactin suggesting that the same pathways are involved. Knockdown of cortactin by siRNA did not inhibit insulin-induced Akt phosphorylation but completely inhibited actin stress fiber formation and glucose uptake. These results suggest that the actin binding protein cortactin is required for actin stress fiber formation in muscle cells and that this process is absolutely required for translocation of GLUT4-containing vesicles to the plasma membrane.

Cell type-specific gene expression in the neuroendocrine system. A neuroendocrine-specific regulatory element in the promoter of chromogranin A, a ubiquitous secretory granule core protein.

The acidic secretory protein chromogranin A universally occurs in amine and peptide hormone and neurotransmitter storage granules throughout the neuroendocrine system. What factors govern the activity of the chromogranin A gene, to yield such a widespread yet neuroendocrine-selective pattern of expression? To address this question, we isolated the mouse chromogranin A gene promoter. The promoter conferred cell type-specific expression in several neuroendocrine cell types (adrenal medullary chromaffin cells, anterior pituitary corticotropes, and anterior pituitary somatolactotropes) but not in control (fibroblast or kidney) cells. In neuroendocrine cells, analysis of promoter deletions established both positive and negative transcriptional regulatory domains. A distal positive domain (-4.8/-2.2 kbp) was discovered, as well as negative (-258/-181 bp) and positive (-147/-61 bp) domains in the proximate promoter. The proximate promoter contained a minimal neuroendocrine-specific element between -77 and -61 bp. Sequence alignment of the mouse promoter with corresponding regions in rat and bovine clones indicated that the mouse sequence shares over 85% homology with rat and 52% with bovine promoters. DNaseI footprinting and electrophoretic gel mobility shift assays demonstrated the presence of nuclear factors in neuroendocrine cells that recognized the proximate promoter. We conclude that the chromogranin A promoter contains both positive and negative domains governing its cell type-specific pattern of transcription, and that a small proximate region of the promoter, containing novel as well as previously described elements, interacts specifically with neuroendocrine nuclear proteins, and is thereby sufficient to ensure widespread neuroendocrine expression of the gene.

A functional cyclic AMP response element plays a crucial role in neuroendocrine cell type-specific expression of the secretory granule protein chromogranin A.

Chromogranin A, a soluble acidic protein, is a ubiquitous component of secretory vesicles throughout the neuroendocrine system. We reported previously the cloning and initial characterization of the mouse chromogranin A gene promoter, which showed that the promoter contains both positive and negative domains and that a proximal promoter spanning nucleotides -147 to +42 bp relative to the transcriptional start site is sufficient for neuroendocrine cell type-specific expression. The current study was undertaken to identify the particular elements within this proximal promoter that control tissue-specific expression. We found that deletion or point mutations in the potential cAMP response element (CRE) site at -68 bp virtually abolished promoter activity specifically in neuroendocrine (PC12 chromaffin or AtT20 corticotrope) cells, with little effect on activity in control (NIH3T3 fibroblast) cells; thus, the CRE box is necessary for neuroendocrine cell type-specific activity of the chromogranin A promoter. Furthermore, the effect of the CRE site is enhanced in the context of intact (wild-type) promoter sequences between -147 and -100 bp. DNase I footprint analysis showed that these regions (including the CRE box) bind nuclear proteins present in both neuroendocrine (AtT20) and control (NIH3T3) cells. In AtT20 cells, electrophoretic mobility shift assays and factor-specific antibody supershifts showed that an oligonucleotide containing the chromogranin A CRE site formed a single, homogeneous protein-DNA complex containing the CRE-binding protein CREB. However, in control NIH3T3 cells we found evidence for an additional immunologically unrelated protein in this complex. A single copy of this oligonucleotide was able to confer neuroendocrine-specific expression to a heterologous (thymidine kinase) promoter, albeit with less fold selectivity than the full proximal chromogranin A promoter. Hence, the CRE site was partially sufficient to explain the neuroendocrine cell type specificity of the promoter. The functional activity of the CRE site was confirmed through studies of the endogenous chromogranin A gene. Northern mRNA analysis showed that expression of the endogenous chromogranin A gene was stimulated seven- to eightfold by cAMP in PC12 cells, whereas no induction occurred in the NIH3T3 cells. Similar cAMP induction was obtained with the transfected chromogranin A promoter in PC12 cells, and abolition of the CRE site (by deletion or point mutation) eliminated the induction. Thus, the CRE site in the chromogranin A proximal promoter is functional and plays a crucial, indeed indispensable, role in neuroendocrine-specific expression of the gene. These results also provide insight into transcriptional mechanisms governing acquisition of the neuroendocrine secretory phenotype.

Amyloid peptides mediate hypoxic increase of L-type Ca2+ channels in central neurones.

Prolonged hypoxia, encountered in individuals suffering from various cardiorespiratory diseases, enhances the likelihood of subsequently developing Alzheimer's disease (AD). However, the underlying mechanisms are unknown, as are the mechanisms of neurodegeneration of amyloid beta peptides (AbetaPs), although the latter involves disruption of Ca2+ homeostasis. Here, immunohistochemistry demonstrated that hypoxia increased production of AbetaPs, an effect which was prevented by inhibition of either beta or gamma secretase, the enzymes required for liberation of AbetaP from its precursor protein. Whole-cell patch clamp recordings showed that hypoxia selectively increased functional expression of L-type Ca2+ channels. This was prevented by inhibition of either beta or gamma secretase, indicating that hypoxic channel up-regulation is dependent upon AbetaP formation. Our results indicate for the first time that hypoxia promotes AbetaP formation in central neurons, and show that this leads to abnormally high selective expression of L-type Ca2+ channels whose blockade has previously been shown to be neuroprotective in AD models. These findings provide a cellular basis for understanding the increased incidence of AD following prolonged hypoxia.

Elevated insulin-like growth factor I receptor autophosphorylation and kinase activity in human breast cancer.

Insulin-like growth factor I action has been implicated in the pathogenesis of many different malignancies, including breast cancer. Insulin-like growth factor I receptors (IGF-IRs) are overexpressed in virtually all breast cancer cell lines, in which they are believed to enhance growth and inhibit apoptosis. In this study, the functional activity of IGF-IRs from normal and malignant human breast tissue was assessed. IGF-IR expression was 14-fold higher in malignant breast tissue than in normal breast tissue. IGF-IR autophosphorylation and kinase activity were 2-4-fold higher in purified receptor preparations from malignant breast tissue as compared to normal breast tissue when normalized for receptor number. This increase in receptor function, coupled with the enhanced receptor expression, amounts to a 40-fold elevation in IGF-IR tyrosine kinase activity in malignant breast tissue. The enhanced receptor autophosphorylation and kinase activity were observed in the absence of hormonal stimulation and seem to result from an alteration in the intrinsic activity of the receptor itself. Protein tyrosine phosphatase activity is also increased in malignant breast tissue. These data suggest that the IGF-IR is an important target for breast cancer therapy.

Repression of the insulin receptor promoter by the tumor suppressor gene product p53: a possible mechanism for receptor overexpression in breast cancer.

There is strong evidence to suggest that insulin and insulin-like growth factor (IGF)-I may be important for tumor growth. Both the insulin and IGF-I receptors (IGF-IR) are overexpressed in breast cancer, and antibody blockade of the IGF-IR inhibits the growth of some breast cancer cell lines. Furthermore, expression of an insulin receptor (IR) in a normal mammary epithelia] cell line causes insulin-dependent transformation. Functional inactivation of p53 is also very frequent in many tumors. In this paper, we investigated whether inactivation of p53 might be involved in the overexpression of the IR in malignancy, specifically breast cancer. We demonstrate a positive correlation between IR and IGF-IR levels and p53 overexpression in primary human breast malignancies. To examine possible mechanisms by which p53 may regulate IR gene expression, we show that p53 can repress the IR promoter and that a dominant-negative p53 (248Q) can de-repress the promoter in cells containing normal p53. The p53 effect was shown to be mediated by C/EBP and Sp1 transcription factors. We also documented that p53-null mice had elevated levels of Sp1, but not C/EBPalpha, and that insulin binding to liver extracts was increased compared to wild-type controls. These results suggest that p53 inactivation may lead to an up-regulation of genes, such as the IR, that are dependent on these transcription factors.

Inhibition of PLC-gamma1 activity converts nerve growth factor from an anti-mitogenic to a mitogenic signal in CHO cells.

Nerve growth factor (NGF) treatment of Chinese hamster ovary fibroblast (CHO) cells exogenously expressing 2.5x105 TrkA receptors (CHO/TrkA) results in inhibition of serum and insulin-like growth factor-I (IGF-I) stimulated cell proliferation in a dose-dependent manner. Furthermore, NGF does not stimulate [3H]thymidine incorporation and inhibits IGF-I mediated DNA synthesis in CHO/TrkA cells. NGF and IGF-I induce extracellular-signal regulated kinase 1 (ERK1) and ERK2 activation, but NGF is able to stimulate a higher and more sustained activation of these enzymes compared with IGF-I. Cotreatment with NGF and IGF-I yields an ERK1/2 activity profile similar to that of NGF treatment alone. While pretreatment with mitogen activated protein kinase kinase (MKK) inhibitor PD98059 (30 microM) results in 100% inhibition of IGF-I stimulated MAPK phosphorylation (IC50<1 microM), NGF mediated MAPK phosphorylation is only decreased by 50% (IC50=3 microM). NGF, but not IGF-I, stimulates tyrosine phosphorylation and activation of PLC-gamma1 which can be inhibited in a dose-dependent manner by phosphoinositide-specific phospholipase C (PI-PLC) inhibitor U73122 (IC50=4 microM). Pretreatment with U73122 (IC50=7 microM) results in an 87% inhibition of NGF mediated MAPK phosphorylation, while cotreatment with PD98059 and U73122 results in 97% inhibition. U73122 pretreatment has no effect on NGF stimulated Akt activation. NGF, but not IGF-I, stimulates the tyrosine phosphorylation of Suc1-associated neurotrophic factor-induced tyrosine phosphorylation target (SNT-1)/fibroblast growth factor receptor substrate 2 (FRS2) which can be completely prevented by pretreatment with 10 microM U73122. Finally, inhibition of PI-PLC results in NGF's ability to stimulate DNA synthesis in the absence and presence of IGF-I.

An upstream element from the human insulin receptor gene promoter contains binding sites for C/EBP beta and NF-1.

We have shown previously that a 500-bp region of the human insulin receptor promoter (-0.3 to -1.8 kb) was able to stimulate transcription from a heterologous thymidine kinase promoter in HepG2 hepatoma cells but not in HeLa fibroblasts. Footprint analysis localized the transcription factor binding sites to a 36-bp region at -1420. In this paper, we analyze the factors that recognize this element and show that it contains binding sites for the CAAT/enhancer binding protein C/EBP and nuclear factor 1 (NF-1). In addition we show that both C/EBP alpha and the C/EBP beta can transactivate the human insulin receptor promoter in a dose-dependent manner.

Stimulation of MAPK cascades by insulin and osmotic shock: lack of an involvement of p38 mitogen-activated protein kinase in glucose transport in 3T3-L1 adipocytes.

Osmotic shock and insulin stimulate GLUT4 translocation and glucose transport via mechanisms that are for the most part distinct yet convergent. In this article, we investigated the effect of osmotic shock and insulin on the activation of the mitogen-activated protein kinase (MAPK) cascades in differentiated 3T3-L1 adipocytes. The MAPKs are activated by phosphorylation on conserved tyrosine and threonine residues. Both sorbitol and insulin strongly stimulated extracellular regulated kinase (ERK) 1 and 2 phosphorylation (8- and 18-fold, respectively). In contrast, c-jun-NH2-terminal kinase (JNK)/stress-activated protein kinase (SAPK) phosphorylation was stimulated only by sorbitol (sevenfold) and not by insulin. Phosphorylation of p38 MAPK was stimulated strongly by sorbitol (22-fold) but weakly by insulin (2.7-fold). Measurement of intrinsic JNK and p38 MAPK activity confirmed the phosphorylation studies. JNK and p38 MAPK were activated only significantly by sorbitol. The MAPKs are phosphorylated by dual-specificity kinases (mitogen-activated ERK-activating kinase [MEK] or MAPK kinase [MKK]). As expected, sorbitol and insulin both stimulated MEK phosphorylation. MKK4 was phosphorylated only in response to sorbitol, and neither of the stimuli caused phosphorylation of MKK3 or 6. To determine the functional significance of the observed activation of p38 MAPK in response to insulin and osmotic shock, we used three pyridinyl imidazole p38 MAPK inhibitors, SB203580, SB202190, and PD169316. Insulin and osmotic shock-stimulated glucose transport was not inhibited by any inhibitor at concentrations that were shown to block p38 MAPK activity. Furthermore, activation of the p38 MAPK pathway by treatment of cells with anisomycin did not stimulate glucose transport. These results suggest that activation of the p38 MAPK pathway is not involved in the stimulation of glucose transport.

Omega-3 fatty acids reduce obesity-induced tumor progression independent of GPR120 in a mouse model of postmenopausal breast cancer.

Obesity and inflammation are both risk factors for a variety of cancers, including breast cancer in postmenopausal women. Intake of omega-3 polyunsaturated fatty acids (ω-3 PUFAs) decreases the risk of breast cancer, and also reduces obesity-associated inflammation and insulin resistance, but whether the two effects are related is currently unknown. We tested this hypothesis in a postmenopausal breast cancer model using ovariectomized, immune-competent female mice orthotopically injected with Py230 mammary tumor cells. Obesity, whether triggered genetically or by high-fat diet (HFD) feeding, increased inflammation in the mammary fat pad and promoted mammary tumorigenesis. The presence of tumor cells in the mammary fat pad further enhanced the local inflammatory milieu. Tumor necrosis factor-alpha (TNF-α) was the most highly upregulated cytokine in the obese mammary fat pad, and we observed that TNF-α dose-dependently stimulated Py230 cell growth in vitro. An ω-3 PUFA-enriched HFD (referred to as fish oil diet, FOD) reduced inflammation in the obese mammary fat pad in the absence of tumor cells and inhibited Py230 tumor growth in vivo. Although some anti-inflammatory effects of ω-3 PUFAs were previously shown to be mediated by the G-protein-coupled receptor 120 (GPR120), the FOD reduced Py230 tumor burden in GPR120-deficient mice to a similar degree as observed in wild-type mice, indicating that the effect of FOD to reduce tumor growth does not require GPR120 in the host mouse. Instead, in vitro studies demonstrated that ω-3 PUFAs act directly on tumor cells to activate c-Jun N-terminal kinase, inhibit proliferation and induce apoptosis. Our results show that obesity promotes mammary tumor progression in this model of postmenopausal breast cancer and that ω-3 PUFAs, independent of GPR120, inhibit mammary tumor progression in obese mice.

Deletion of the insulin receptor beta-subunit acidic domain results in enhanced metabolic signaling.

The contribution of the insulin receptor beta-subunit acidic domain, amino acids 1262-1291, to receptor function was analyzed. A mutant insulin receptor complementary DNA lacking this domain was created. Rat-1 fibroblasts were stably transfected with plasmids containing the mutant insulin receptor complementary DNA and clonal cell lines derived (hIR1262). Compared with cells overexpressing the wild type insulin receptor, metabolic signaling was enhanced in hIR1262 cells whereas the mitogenic response to insulin was unchanged. hIR1262 had normal kinase activity and insulin-stimulated receptor internalization in spite of substantially reduced autophosphorylation (70% decreased in vitro). Additionally, polylysine, a polycation postulated to interact with the insulin receptor beta-subunit acidic domain, increased autophosphorylation and facilitated insulin-induced phosphorylation of calmodulin in the wild type as well as the hIR1262 receptors. We conclude: 1) The acidic domain is not the site of interaction between the insulin receptor and polycations. 2) Removal of the acidic domain leads to enhanced metabolic signaling but 3) unchanged mitogenic activity. 4) The defect in autophosphorylation is not correlated with a defect in kinase activity. Thus, the observed changes in biological signaling indicate that specific pathways diverge at the level of the receptor itself and that neither kinase activity or biological activity necessarily correlates directly with diminished autophosphorylation when the tyrosine kinase domain remains intact.

Relative expression of insulin receptor isoforms does not differ in lean, obese, and noninsulin-dependent diabetes mellitus subjects.

The insulin receptor is expressed as two isoforms that differ by a 12-amino acid region at the carboxy-terminus of the alpha-subunit encoded by exon 11. These isoforms are produced by tissue-specific alternate splicing of the insulin receptor mRNA. To determine whether the relative expression of the isoforms is altered in skeletal muscle in two insulin-resistant states, NIDDM and obesity, relative mRNA levels were measured using a polymerase chain reaction technique. There were no differences in the relative amounts of skeletal muscle mRNA encoding the exon 11-containing form compared to the exon 11-lacking form of the insulin receptor among lean normal (30 +/- 2% Ex11-), obese nondiabetic (32 +/- 2%), and NIDDM (31 +/- 1%) subjects. We conclude that altered expression of insulin receptor isoform mRNAs does not account for skeletal muscle insulin resistance in NIDDM and obesity.

Altered insulin receptor messenger ribonucleic acid splicing in liver is associated with deterioration of glucose tolerance in the spontaneously obese and diabetic rhesus monkey: analysis of controversy between monkey and human studies.

There are two insulin receptor (IR) isoforms (designated type A and type B), derived from alternative splicing of exon 11 of the IR gene. Recently, we reported (Huang Z., Bodkin N.L., Ortmeyer H.K., Hansen B.C., Shuldiner A. R., 1994, J Clin Invest, 94:1289-1296) that an increase in the exon 11- (i.e. lacking exon 11) (type A) IR messenger RNA (mRNA) variant in muscle is associated with hyperinsulinemia, an early risk factor for noninsulin-dependent diabetes mellitus (NIDDM), in the spontaneously obese, diabetic rhesus monkey. To explore further the role of IR mRNA splicing in insulin resistance of NIDDM, we studied liver, another target organ that is resistant to insulin action in NIDDM. The relative amounts of the two IR mRNA-splicing variants in liver were quantitated by RT-PCR in normal, prediabetic, and diabetic (NIDDM) monkeys. The percentage of the exon 11- mRNA variant in liver (n = 24) was significantly correlated with fasting plasma glucose (r = 0.55, P < 0.01) and intravenous glucose disappearance rate (r = -0.45, P < 0.05). The exon 11- mRNA variant was increased significantly from 29.8 +/- 1.6% in monkeys with normal fasting glucose to 39.2 +/- 2.9% in monkeys with elevated fasting glucose (P < 0.01). These studies provide the first direct evidence in vivo that the relative expression of the two IR mRNA-splicing variants is altered in liver and suggest that increased expression of the exon 11- IR isoform may contribute to hepatic insulin resistance and NIDDM or may compensate for some yet unidentified defect.

Hypoxic enhancement of evoked noradrenaline release from the human neuroblastoma SH-SY5Y.

The effects of chronic hypoxia (2.5% O(2), 24 h) on [3H]noradrenaline ([3H]NA) release evoked from human neuroblastoma SH-SY5Y cells by depolarisation and by activation of muscarinic receptors was investigated. Depolarization of cells with 100 mM K(+) evoked [3H]NA release, and chronic hypoxia enhanced this release significantly. In fluorimetric studies, the K(+)-evoked rises of [Ca(2+)](i) observed in response to 100 mM K(+) were also significantly enhanced. Muscarine-evoked [3H]NA release was also dramatically enhanced by chronic hypoxia. However, muscarine-induced release of Ca(2+) from intracellular stores and subsequent capacitative Ca(2+) entry was unaffected. The protein kinase C inhibitors GF 109 203X and RO-31-8220 did not prevent the enhancement of muscarine-evoked release caused by chronic hypoxia. These findings indicate that chronic hypoxia increases release of [3H]NA from human neuroblastoma SH-SY5Y cells. Enhancement of K(+)-evoked release was attributable to an enhancement of depolarisation-mediated Ca(2+) influx. In contrast, the larger enhancement of muscarine-evoked [3H]NA release was not due to greater release of Ca(2+) from internal stores, nor due to enhanced Ca(2+) influx. Furthermore, it was not attributable to activation of protein kinase C. These findings suggest that enhancement of sympathetic output, known to occur following prolonged hypoxia, may be mediated in part by enhancement of exocytosis.

Inhibition of depolarisation-evoked [(3)H]noradrenaline release from SH-SYFY human neuroblastoma cells by muscarinic (M1) receptors is not mediated by changes in [Ca(2+)].

The aim of this study was to obtain further understanding of the mechanism by which activation of muscarinic M(1) receptors inhibits K(+)-evoked noradrenaline (NA) release in the human neuroblastoma SH-SY5Y. Previous studies have found that muscarinic M(1) and M(3) receptors couple to the activation of phospholipase C in SH-SY5Y cells leading to an increase in (a) intracellular calcium ([Ca(2+)](i)) and (b) activation of protein kinase C (PKC). This study used specific inhibitors of PKC and conditions which deplete Ca(2+)(i) stores to examine the role of protein kinase C and changes in [Ca(2+)](i) in mediating the inhibition of K(+)-evoked NA release by muscarine. Our data show that pretreatment of SH-SY5Y cell layers with bisindolylmaleimide I (BIM-I) (i) failed to reverse inhibition of K(+)-evoked NA release by muscarine but (ii) did overcome the attenuation of muscarine inhibition following pretreatment with TPA. Furthermore pretreating cell layers with Ca(2+)-free Hepes buffered saline in the presence of thapsigargin, conditions which prevented muscarine induced increases in [Ca(2+)](i), failed to prevent inhibition of K(+)-evoked NA release by muscarine. The effect of muscarine on K(+)-evoked uptake of Ca(2+)(e) was examined in SH-SY5Y cells loaded with Fura-2. Muscarine inhibited Ca(2+)(e)-uptake by decreasing the rate at which Ca(2+) entered SH-SY5Y cells via voltage sensitive Ca(2+)-channels. Thus this study shows that muscarine inhibits depolarisation-evoked NA release by a mechanism which is not dependent on activation of PKC or release of Ca(2+) from internal stores.

Induction of insulin resistance in human skeletal muscle cells by downregulation of glycogen synthase protein expression.

Glycogen synthase (GS) is the rate-limiting enzyme controlling nonoxidative glucose disposal in skeletal muscle. A reduction in GS activity and an impaired insulin responsiveness are characteristic features of skeletal muscle in type 2 diabetes. These properties also exist in human skeletal muscle cell cultures from type 2 diabetic subjects. To determine the effect of an isolated reduction in GS on skeletal muscle insulin action, cultures from nondiabetic subjects were treated with antisense oligonucleotides (ODNs) to GS to interfere with expression of the gene. Treatment with antisense ODNs reduced GS protein expression by 70% compared with control (scrambled) ODNs (P < .01). GS activity measured at 0.01 mmol/L glucose-6-phosphate (G-6-P) was reduced by antisense ODN treatment. The insulin responsiveness of GS was impaired. Insulin also failed to stimulate glucose incorporation into glycogen after antisense ODN treatment. The cellular glycogen content was lower in antisense ODN-treated cells compared with control ODN. The insulin responsiveness of glucose uptake was abolished by antisense ODN treatment. Thus, reductions in GS expression in human skeletal muscle cells lead to impairments in insulin responsiveness and may play an important role in insulin-resistant states.

Effect of dexamethasone on the alternative splicing of the insulin receptor mRNA and insulin action in HepG2 hepatoma cells.

We have shown that culturing HepG2 cells in Ham's F-12 medium supplemented with calf serum, dexamethasone, and triiodothyronine causes an increase in the insulin sensitivity and responsiveness for glucose incorporation into glycogen. This correlates with increased expression of the mRNA encoding the B isoform of the insulin receptor. Of all the components in the medium, we found that dexamethasone exerted the greatest effect. Dexamethasone alone could cause both a switch in expression from the A to the B isoform of the insulin receptor and also an increase in insulin sensitivity for both glucose incorporation into glycogen and 2-deoxyglucose transport. In addition, we found that expression of the B isoform of the insulin receptor is developmentally regulated in the 3T3-L1 adipocyte leading to the suggestion that the alternatively spliced B isoform of the insulin receptor may play an important role in signal transduction in insulin target tissues such as liver, fat, and muscle.

Mutation of the two carboxyl-terminal tyrosines results in an insulin receptor with normal metabolic signaling but enhanced mitogenic signaling properties.

Our previous studies have shown that the deletion of the insulin receptor carboxyl terminus impairs metabolic, but augments mitogenic, signaling (McClain, D. A., Maegawa, H., Levy, J., Huecksteadt, T., Dull, T. J., Lee, J., Ullrich, A., and Olefsky, J. M. (1988) J. Biol. Chem. 263, 8904-8911; Thies, R.S., Ulrich, A., and McClain, D. A. (1989) J. Biol. Chem. 264, 12820-12825). To explore further the regulatory role of the insulin receptor carboxyl terminus, a mutant insulin receptor was constructed in which the two tyrosines (Y1316 and Y1322) on the carboxyl terminus were replaced with phenylalanines. Rat 1 fibroblasts expressing high levels of this mutant receptor (Y/F2 cells) exhibited normal insulin binding and normal insulin internalization. The absence of the two tyrosines in the carboxyl terminus did not affect the phosphotransferase activity of the beta-subunit and insulin-stimulated glucose transport. However, the Y/F2 cells showed markedly enhanced sensitivity for insulin-stimulated DNA synthesis. Dose-response curves for both insulin-stimulated thymidine uptake and 5-bromo-2-deoxyuridine incorporation in the Y/F2 cell lines were shifted to the left (4-10-fold) compared with those observed in the cells expressing similar numbers of wild type receptors. Thus, the two tyrosines of the insulin receptor carboxyl terminus do not modulate the kinase function of the insulin receptor, although they are autophosphorylated in native receptors. Moreover, these tyrosines are not necessary for stimulation of glucose transport. On the other hand, these results suggest that the two carboxyl-terminal tyrosine residues exert an inhibitory effect on mitogenic signaling in native insulin receptors.

Starvation and refeeding regulate glycogen synthase gene expression in rat liver at the posttranscriptional level.

Starvation and refeeding affect glycogen metabolism. The effects of starvation and refeeding on the level of glycogen synthase (GS) gene expression were examined in rat liver. Depletion of hepatic glycogen stores by 72 h of starvation (7% of control) was supercompensated by 24 h of refeeding a standard laboratory diet (247% of control). Upon further refeeding, glycogen concentration gradually returned to control levels after 120 h. After 72 h of starvation, GS activity and immunoreactive protein in the liver were 60-64% lower than in control rats with free access to food. After 72 h of refeeding, GS activity and immunoreactive protein returned to control values. No significant differences in GS mRNA levels were found between fed, starved and refed rats, as determined by Northern blot analysis and PCR quantification, indicating that the long-term regulation of GS gene expression in starvation and refeeding occurs via a posttranscriptional mechanism. The amount of GS mRNA associated with polyribosomes was 90% lower in starved than in fed rats. These data indicate that the efficiency of GS mRNA translation, rather than its abundance, decreases during starvation.