Abstract: Serum and glucocorticoid regulated kinase 1 (Sgk1) is a serine-threonine kinase that is activated by serum, steroids, insulin, vasopressin, and interleukin 2 at the transcriptional and post-translational levels. Sgk1 is also important in transduction of growth factors and steroid-dependent survival signals and may have a role in the development of resistance to cancer chemotherapy. In the present paper, we demonstrate that Sgk1 activates MDM2-dependent p53 ubiquitylation. The results were obtained in RKO cells and other cell lines by Sgk1-specific RNA silencing and were corroborated in an original mouse model as well as in transiently and in stably transfected HeLa cells expressing wild-type or dominant negative Sgk1 mutant. Sgk1 contributes to cell survival, cell-cycle progression, and epithelial de-differentiation. We also show that the effects of Sgk1 on the clonogenic potential of different cancer cells depend on the expression of wild-type p53. Since transcription of Sgk1 is activated by p53, we propose a finely tuned feedback model where Sgk1 down-regulates the expression of p53 by enhancing its mono- and polyubiquitylation.

Abstract: The serum and glucocorticoid inducible kinase SGK1 was originally cloned from mammary tumor cells. SGK1 was found to be up-regulated in a variety of tumors, but down-regulated in several distinct tumors. Thus, evidence for a role of SGK1 in tumor growth remained conflicting. According to in vitro observations, SGK1 is up-regulated by the oncogene beta-catenin and negatively regulates the proapoptotic transcription factor FOXO3a, which in turn stimulates transcription of the Bcl2-interacting mediator BIM. This study aimed to define the role of SGK1 in colon carcinoma in vivo. SGK1 knockout mice (sgk1(-/-)) and their wild type littermates (sgk1(+/+)) were subjected to chemical cancerogenesis (intraperitoneal injection of 20 mg/kg 1,2-dimethylhydrazine followed by three cycles of 30 g/L synthetic dextran sulfate sodium for 7 days). Moreover, SGK1 was silenced in HEK293 cells. FOXO3a and BIM protein abundance was determined by Western blotting and immunohistochemistry. Following chemical cancerogenesis, sgk1(-/-)mice developed significantly less colonic tumors than sgk1(+/+)mice. According to Western blotting and immunohistochemistry, SGK1 deficiency enhanced the expression of FOXO3a and BIM both, in vitro and in vivo. SGK1 deficiency counteracts the development of colonic tumors, an effect at least in part due to up-regulation of FOXO3a and BIM.

Abstract: The interleukin-2 is a cytokine that is essential for lymphocytic survival and function. Ectopic expression of the IL-2 receptor in epithelial tissues has been reported previously, although the functional significance of this expression is still being investigated. We provided novel structural and functional information on the expression of the IL-2 receptor in kidney cancer cells and in other normal and neoplastic human epithelial tissues. In A-498 kidney cancer cells, we showed that IL-2 binding to its own receptor triggers a signal transduction pathway leading to the inhibition of proliferation and apoptosis. We found that the inhibition of proliferation is associated with Erk1/2 dephosphorylation, whereas the survival signals appear to be mediated by Sgk1 activation. This investigation focuses on the IL-2 induced regulation of Sgk1 and describes a role of the IL-2 receptor and Sgk1 in the regulation of epithelial tumor cell death and survival.

Abstract: Mutations in the gene (GJB2) coding for Connexin 26 (Cx26) are responsible for genetic forms of sensorineural hearing loss. This article describes a family characterized by congenital profound hearing loss, inherited in an autosomal dominant fashion and associated to a R75Q substitution in Cx26. Cell transfection and fluorescence imaging, dye transfer experiments and dual patch clamp recording showed that the mutant completely prevents the formation of functional channels despite assembling into junctional plaques, in communication incompetent HeLa cells. The disease is not associated with palmar and plantar keratosis in any of the family members, suggesting that R75Q substitution is not sufficient for the development of the complete syndromic phenotype. The association of palmar and plantar keratosis with profound hearing loss may be dependent on genetic background, requiring a functional interaction between the mutated Cx26 and other epidermally expressed connexins.

Abstract: Serum- and glucocorticoid-regulated kinase (Sgk1) is considered to be an essential convergence point for peptide and steroid regulation of ENaC-mediated sodium transport. We tried to identify molecular partners of Sgk1 by yeast two-hybrid screening. Yeast two-hybrid screening showed a specific interaction between Sgk1 and phosphomannomutase (PMM)2, the latter of which is an enzyme involved in the regulation of glycoprotein biosynthesis. The interaction was confirmed in intact cells by coimmunoprecipitation and colocalization detected using confocal microscopy. We were then able to demonstrate that Sgk1 phosphorylated PMM2 in an in vitro assay. In addition, we found that the enzymatic activity of PMM2 is upregulated by insulin treatment and that Sgk1 completely inhibits PMM2 activity both in the absence and in the presence of insulin stimulation. These data provide evidence suggesting that Sgk1 may modulate insulin action on the cotranslational glycosylation of glycoproteins.

Abstract: Adducin regulates tubular absorption of sodium by modulating the expression levels of the sodium-potassium-ATPase in renal tubular cells. Adducin is a candidate gene in the pathogenesis of hypertension. Yeast two hybrid screen showed a specific interaction between human alpha Adducin and the regulatory factor for X box (RFX-1), a nuclear protein that down regulates the expression of several proteins in non neuronal cells. The interaction was confirmed in cells through co-immunoprecipitation and colocalization experiments. The binding of alpha Adducin to RFX-I and their nuclear co-localization suggests that Adducin can have a role in modulating the transcriptional regulating activity of RFX-I.

Abstract: To study the role of sgk (serum, glucocorticoid-induced kinase) in hormonal regulation of Na+ transport mediated by the epithelial Na+ channel (ENaC), clonal cell lines stably expressing human sgk, an S422A sgk mutant, or a D222A sgk mutant were created in the background of the A6 model renal epithelial cell line. Expression of normal sgk results in a 3.5-fold enhancement of basal transport and potentiation of the natriferic response to antidiuretic hormone (ADH). Transfection of a S422A mutant form of sgk, which cannot be phosphorylated by phosphatidylinositol-dependent kinase (PDK)-2, results in a cell line that is indistinguishable from the parent line in basal and hormone-stimulated Na+ transport. The D222A sgk mutant, which lacks kinase activity, functions as a dominant-negative mutant inhibiting basal as well as peptide- and steroid hormone-stimulated Na+ transport. Thus sgk activity is necessary for ENaC-mediated Na+ transport. Phosphorylation and activation by PDK-2 are necessary for sgk stimulation of ENaC. Expression of normal sgk over endogenous levels results in a potentiated natriferic response to ADH, suggesting that the enzyme is a rate-limiting step for the hormone response. In contrast, sgk does not appear to be the rate-limiting step for the cellular response to aldosterone or insulin.

Abstract: BACKGROUND: Reactive oxygen species play a critical role in inducing apoptosis. The small GTPase p21 Ras and the ERK1/2 MAPK have been proposed as key regulators of the signaling cascade triggered by oxidative stress (H2O2). Harvey-Ras (Ha-Ras) and Kirsten-Ras (Ki-Ras) isoforms are so far functionally indistinguishable, because they activate the same downstream effectors, including ERK1/2. Moreover, ERK1/2 signaling has been involved in both protection and induction of apoptosis. METHODS AND RESULTS: Human umbilical vein endothelial cells (HUVECs) were subjected to H2O2, and apoptosis was detected by fluorescence-activated cell sorting analysis, fluorescence microscopy, and caspase-3 activation. Transfection of Ha-Ras and Ki-Ras genes in HUVECs was performed to evaluate the response to H2O2. We have found that, whereas Ha-Ras decreases tolerance to oxidative stress, Ki-Ras has a potent antiapoptotic activity. Both effects are mediated by ERK1/2. Tolerance to H2O2 is encoded by a unique stretch of lysines at the COOH terminus of the Ki-Ras, lacking in Ha-Ras, and it is relatively independent of the farnesylated anchor. Inhibition of p21 Ras signaling by farnesylation inhibitors increased the resistance to apoptosis in Ha-Ras-expressing cells. CONCLUSIONS: These findings explain the opposite effects of ERK1/2 stimulation on apoptosis found in different cell types and suggest that local activation of ERK1/2 signaling may account for the opposing response to oxidative stress by Ha-Ras or Ki-Ras-expressing cells. Modulation of cell reactivity to oxidative stress by p21 Ras points to the specific and predictive effects of Ras inhibitors in vivo as potential therapeutic drugs in disorders produced by increase of reactive oxygen species inside the cells.

Abstract: Sgk (serum- and glucocorticoid-induced protein kinase) is a serine/threonine-specific protein kinase that is transcriptionally regulated by serum, glucorticoids, and mineralocorticoids. Sgk regulates the amiloride-sensitive sodium channel in kidney principal cells. Insulin and insulin-like growth factor-1 stimulate activity of Sgk by a mechanism mediated by phosphoinositide-dependent kinases (PDK)-1 and -2. In this study, we demonstrate that incubation of transfected cells with 8-(4-chlorophenylthio)-cAMP (8CPT-cAMP; 0.2 mm) led to a 2-fold activation of recombinant Sgk expressed in COS7 cells. Furthermore, the combination of insulin plus 8CPT-cAMP elicited a larger response than either agent alone. The effect of insulin was inhibited by wortmannin (100 nm), but not by the cyclic AMP-dependent protein kinase (PKA) inhibitor, H89 (10 microm). As expected, the effect of 8CPT-cAMP was completely blocked by H89. Surprisingly, the effect of 8CPT-cAMP was also inhibited by wortmannin, suggesting that phosphorylation of Sgk by PDK-1 and/or -2 is required for activation by 8CPT-cAMP. Mutational analysis led to similar conclusions. The Thr(369) --> Ala mutant, lacking the PKA phosphorylation site, was activated by insulin but not 8CPT-cAMP. In contrast, the Ser(422) --> Ala mutant, lacking a PDK-2 phosphorylation site, was inactive and resistant to activation by either insulin or 8CPT-cAMP. In summary, Sgk is subject to complex regulatory mechanisms. In addition to regulation at the level of gene expression, the enzymatic activity of Sgk is regulated by multiple protein kinases, including PKA, PDK-1, and PDK-2. Cross-talk among these signaling pathways may play an important role in the pathogenesis of the hypertension associated with hyperinsulinemia, obesity, and insulin resistance.

Abstract: The association between angiotensin-converting enzyme (ACE) gene polymorphism and insulin resistance (IR) in hypertensive subjects remains controversial. Thus, we evaluated the possible association between IR and ACE gene polymorphism in a group of hypertensive, never-treated patients compared with that in a normotensive control group. We enrolled 200 (114 men and 86 women; age, 45.5 +/- 4.7 yr) hypertensive patients and 96 (54 men and 42 women; age, 44.0 +/- 4.7 yr) normotensive subjects. A double PCR assay was used to identify ACE genotypes. We determined fasting glucose and insulin by the glucose oxidase method and using a standard RIA technique. IR was estimated using the homeostasis model assessment (HOMA(IR)). Both fasting glucose (5.0 +/- 0.3 vs. 4.7 +/- 0.3 mmol/L; P < 0.0001), insulin levels (12.3 +/- 4.7 vs. 4.9 +/- 1.5 muU/mL; P < 0.0001), and HOMA(IR) (2.7 +/- 1.1 vs. 1.1 +/- 0.3; P < 0.0001) were significantly higher in hypertensive patients than in the normotensive control group. When we subdivided hypertensive patients according to ACE genotype, we observed that fasting insulin and HOMA(IR) were 16.3 +/- 3.3 and 3.6 +/- 0.8 in the DD genotype, 9.4 +/- 3.1 and 2.1 +/- 0.7 in the ID genotype, and 8.3 +/- 2.8 and 1.9 +/- 0.7 muU/mL in the II group (P < 0.0001, by ANOVA). No significant differences were observed in the normotensive control group. In conclusion, we extended previous data regarding the relationship of hypertension and IR by demonstrating a dependence of this relationship upon the ACE gene polymorphism.

Abstract: The response of the forearm vasculature to acetylcholine (7.5, 15, and 30 microg/min, each for 5 minutes) and sodium nitroprusside (0.8, 1.6, and 3.2 microg/min, each for 5 minutes) was evaluated in 32 never-treated hypertensive outpatients (17 men and 15 women, aged 43+/-7 years) and in 24 normotensive control subjects (14 men and 10 women, aged 42+/-6 years). Drugs were infused into the brachial artery, and forearm blood flow was measured by strain-gauge plethysmography. In both hypertensive and normotensive groups, a deletion (D)/insertion (I) polymorphism in intron 16 of the angiotensin-converting enzyme (ACE) gene was determined by polymerase chain reaction. The response to acetylcholine was significantly reduced in hypertensive patients versus control subjects: at the highest dose (30 microg/min), forearm blood flow was 13.9+/-6.3 mL x 100 mL tissue(-1) x min(-1) in hypertensives versus 27.1+/-9.7 mL x 100 mL tissue(-1) x min(-1) in the controls (P<.001); similarly, vascular resistance was 10.6+/-5.6 U in hypertensive patients and 4.9+/-1.9 U in normotensive subjects. In the hypertensive group, the patients with DD genotype showed significantly less endothelium-dependent vasodilation compared with ID+II genotypes (at the highest dose of acetylcholine, forearm blood flow was 12.1+/-4.2 versus 17.0+/-4.1 mL x 100 mL tissue(-1) x min(-1)) (P<.005). The vasodilator effect of sodium nitroprusside infusions was not statistically different in DD and ID+II hypertensive patients. In conclusion, our data suggest that ACE polymorphism affects endothelium-dependent vasodilation in hypertensive patients and confirm that hypertensive patients had a blunted response to the endothelium-dependent agent acetylcholine.

Abstract: The impact of molecular genetics in the diagnosis and management of various forms of heritable cardiac or vascular disorders is continuously increasing thanks to the newly available laboratory tools. Familial hypertrophic cardiomyopathy (FHC), an autosomal dominant inherited disease characterized by unexplained left ventricular hypertrophy and a wide range of clinical symptoms, is the first cardiac disorder whose genetic bases have been elucidated. Linkage analysis studies have shown a statistically significant association between the disease status and at least seven genetic loci, all coding for sarcomeric proteins, in unrelated kindreds. A major challenge for physicians is to make an accurate and early diagnosis, not only on the basis of the traditional tools (i.e. physical examination and electro-echocardiography) but also to focus on the impact of genotype on clinical manifestations of FHC. In this review we present the more recent findings on the genetic basis of FHC and analyze the genotype-phenotype correlations of this disorder, whose expression may be modulated by additional factors (modifier genes, genetic background, environmental factors) other than mutations in any of the sarcometric proteins.

Abstract: We report a PCR-based technique for detecting thyroid cancer metastases in small nodes <1.5 cm diameter by the amplification of thyroid specific transcripts TSH-receptor and thyroglobulin. A 100% correspondence with the histopathological diagnosis was observed in the 41/46 nodes (89%) in which an adequate sample was obtained at fine needle aspiration. The genetic analysis resulted more sensitive and accurate than both the cytological analysis (28% inadequate samples, 17% false negative diagnoses) and the thyroglobulin measurement in the aspirates (39% false negatives). The PCR-based genetic analysis may provide a useful tool for diagnosis and follow-up of thyroid cancer.

Abstract: OBJECTIVES: This study sought to evaluate the possible association of polymorphism of the angiotensin-converting enzyme (ACE) gene with blood pressure and left ventricular mass index (LVMI). BACKGROUND: The renin-angiotensin system seems to be involved in the pathogenesis of essential hypertension. Moreover, recent epidemiologic observations demonstrate that many subjects with left ventricular hypertrophy have normal blood pressure levels, suggesting that factors other than hemodynamic overload may contribute to the hypertrophy. METHODS: The study included 140 untreated hypertensive outpatients who underwent ambulatory blood pressure monitoring, echocardiographic evaluation and analysis for insertion (I)/ deletion (D) polymorphism in intron 16 of the ACE gene by polymerase chain reaction. Blood pressure was measured at 24 h, and LVMI was calculated by the Devereux formula, in each patient. RESULTS: Left ventricular mass index values (mean +/- SD) were 137 +/- 28 g/m2 in patients with the DD genotype, 125 +/- 27 g/m2 in those with the ID genotype and 115 +/- 27 g/m2 in those with II genotype. The frequencies of the DD, ID and II genotypes were 45.71% (n = 64), 46.42% (n = 65) and 7.85% (n = 11), respectively, and were in Hardy-Weinberg equilibrium. The strongest association between left ventricular mass and DD genotype in our cohort appeared to be an independent cardiovascular risk factor (DD vs. ID: odds ratio [OR] 2.497, 95% confidence interval [CI] interval 1.158 to 5.412, p < 0.05; DD vs. II: OR 6.577, 95% CI 1.169 to 28.580, p < 0.02). CONCLUSIONS: Our data show that the LVMI was significantly enhanced in patients with the DD genotype.

Abstract: The present work describes the distribution of HCV genotypes in Calabria. The data presented suggest that, in the sample of population investigated, genotype 1b is the most prevalent followed by the 2b and the 2a.. In addition it is important to note that in Calabria the prevalence of genotype 1b is strikingly high in respect to the other Italian pullulation. An Association between HCV type 1b and the more severe clinical course of the liver disease has been reported. Although the data presented indicate that in Calabria most of the subjects enrolled in the study are infected by a virulent HCV strain, no association has been found with more severe clinical manifestations.

Abstract: A previously undescribed de novo insertion-deletion mutation in the beta cardiac myosin heavy chain gene was found in a kindred with familial hypertrophic cardiomyopathy. In the mutated allele there is an inserted-deleted guanine at nucleotides 8823 and 8850 of the beta myosin heavy chain gene, resulting in a dramatic change of the amino acid sequence (AA 395-404). such a mutation, detected in the proband and in his son but not in the proband's parents, is likely to produce major impairment of myosin function.

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Abstract: The ACE gene has recently been shown to be associated with myocardial infarction, especially in subjects considered at low risk for coronary heart disease (CHD) according to common classification criteria. The possible relationship between deletion polymorphism in this gene and CHD risk factors, as well as asymptomatic extracoronary atherosclerosis, has been investigated in the present study. One hundred and seventy-four subjects, enrolled in a cardiovascular disease prevention study, underwent clinical and biochemical examination and ACE-I/D polymorphism determination. Subjects > 45 years of age (n = 107) also received echo-Doppler examination of the carotid arteries. Based on the results of ACE-I/D polymorphism, subjects were divided into three groups: homozygous for deletion (D/D), homozygous for insertion (I/I) and heterozygous (I/D). The prevalence of CHD risk factors as well as of extracoronary atherosclerosis was similar in the three genotype groups. Similarly, there was no association between the presence of atherosclerotic lesions and genotype in subjects at low and high CHD risk. Ten subjects with diabetes mellitus had ACE-D/D genotype. Among these subjects seven had hypertension. Eight subjects with diabetes mellitus had ACE-I/D genotype and only one of these was hypertensive. None of the ACE-I/I subjects was diabetic. ACE-I/D polymorphism seems to play a role in the development of hypertension, at least in diabetic subjects. Its determination may help to identify and monitor diabetic subjects prone to hypertension.

Abstract: In the course of studies to elucidate the complex network of interactions controlling FRTL5 cell proliferation, thyroid stimulating hormone (TSH)-independent mutants (M cells), have been obtained from FRTL5 cells by chemical mutagenesis. In the present studies, the role of TSH on the proliferation and on differentiated and metabolic functions in these mutant cells have been investigated and compared to their response to insulin-like growth factor I (IGF-I). The addition of IGF-I to M cells leads to normal stimulation of DNA synthesis. However, inspite of the fact that mutant cells display normal TSH receptors, TSH is unable to stimulate the proliferation of the M cells. Nevertheless, TSH is able to increase intracellular levels of cAMP leading to regulation of TSH function in the M cells. On the other hand, TSH does not influence iodide transport and actin filaments depolimerization in these cells. However, aminoacid transport, stimulated in wild-type FRTL5 cells by both TSH and IGFs, is under the control of IGFs but not of TSH in the mutant cells. Neither TSH or IGF-I modified the expression of c-fos proto-oncogene in the M cells, probably because of high constitutive expression. These data suggest that a crucial signalling step(s) required for TSH induced mitogenesis is impaired in the M cells, and that this signalling step is not required for IGF-I induced mitogenesis.

Abstract: Rat thyroid cells (FRTL5), transfected with the sequence coding for rat insulin-like growth factor II (IGF-II) presented mRNA specific for the transfected IGF-II in most of the clones obtained (Tr clones). Tr7 and Tr12 cells maintained their ability to respond to the mitogenic effect of thyrotropin (TSH), while either exogenous IGF-I or IGF-II or insulin failed to stimulate their proliferation. In the absence of exogenous mitogens the Tr7 and Tr12 clones vigorously incorporated [3H]thymidine into DNA. This activity was significantly inhibited by sm1.2, a monoclonal antibody against rat IGF-II. Tr7 and Tr12 clones possess type I IGF receptors, known to mediate the mitogenic effect of IGF-II, with affinity similar to those present on the membrane of the parental cells but with reduced capacity. Finally, media conditioned by Tr7 and Tr12 increase basal thymidine incorporation in quiescent FRTL5 cells and amplify that induced by TSH. Endogenous IGFs may play an important role in the regulation of thyroid cell proliferation by modulating the mitogenic effect of TSH and by supporting TSH-independent growth.

Abstract: FRTL-5 rat thyroid cells have receptors for both insulin and IGF-I which can be distinguished in binding studies. The ability of TSH to regulate each in an antiparallel manner is atypical. If these receptors are shown to have independent as well as coordinate activities, studies of the mechanisms of their receptor cross-talk in these cells will be relevant to understanding IGF-I and insulin receptors in other tissues.

Abstract: A tumor surface protein (TSP-180) has been identified on murine lung carcinomas using two monoclonal antibodies (MoAbs) (135-13C and 346-11A). Quantitative analysis of TSP-180 on 3LL variants maintained either in vitro or in vivo indicates that TSP-180 is highly expressed in highly malignant metastatic cells. In reducing conditions, sodium dodecyl sulfate-polyacrylamide gel electrophoresis banding patterns of TSP-180 obtained with MoAb 135-13C from cell lysates of 3LL metastatic cells show three proteins migrating to Mr 204,000, 134,000, and 116,000. In the same experimental conditions MoAb 135-13C precipitates from low metastasizing ones only one band, corresponding to the lower molecular weight (Mr 116,000). All bands of TSP-180 observed in 3LL variants are labeled by lactoperoxidase-catalyzed radioiodination of viable cells, incorporate 32PO4, and contain carbohydrates, as judged by binding to wheat germ agglutinin. These results indicate that all proteins have external exposure on the cell surface and that at least some of TSP-180 proteins could be differentially regulated in different tumor cells (highly metastatic versus low metastatic). Sodium dodecyl sulfate-polyacrylamide gel electrophoresis banding patterns and immunoblots obtained from cell lysates of 3LL variants by using a monoclonal antibody to phosphotyrosine (IG-2) indicate that this MoAb recognizes proteins migrating with molecular weights identical to those reported for TSP-180. Moreover, the immunoblots of solubilized immunocomplex, obtained from cell lysates of 3LL variants by using MoAb 135-13C, demonstrate that MoAb IG-2 specifically reacts with TSP-180 proteins. Experiments undertaken in order to assess if some or all of TSP-180 proteins have tyrosine kinase activity demonstrate that MoAb 135-13C binding to the cell surface induces specific phosphorylation of the Mr 204,000 protein of TSP-180. Phosphoaminoacid analysis of the ligand-induced phosphorylated protein (pp204) demonstrates that this protein is phosphorylated at serine and tyrosine. Results reported lead us to hypothesize that TSP-180 is involved in growth-regulation mechanisms and that its high expression on cells with more malignant phenotype could be responsible for a proliferative advantage of such tumor clones.

Abstract: Receptors for Insulin, Epidermal Growth Factor, Platelet-Derived Growth Factor and Insulin-like Growth Factor type 1 are tyrosine-specific protein kinases. This enzymatic activity may play a role in mediating the biological actions of these peptides. It has recently been identified a Mr 120 KDa glycoprotein in rat liver plasma membranes which can be phosphorylated by the insulin receptor and by the EGF receptor in a cell-free system and by the insulin receptor in intact cultured H-35 hepatoma cells. In the present report it is shown that the solubilized Insulin-like Growth Factor type 1 receptor can phosphorylate tyrosine residues in the same 120 KDa glycoprotein from the AS-30D rat hepatoma cells.

Abstract: A tumor surface protein (TSP-180) that is highly expressed on highly malignant metastatic cells has been identified on murine lung carcinomas. On SDS-PAGE under reducing conditions, TSP-180 shows a complex banding pattern corresponding to 204, 183, 150, 135, and 116 kDa. All bands of the TSP-180 complex are glycosylated and are labeled by lactoperoxidase-catalyzed radioiodination of viable cells. The mouse TSP-180 complex described here is homologous to the human integrin alpha 6 beta 4 complex, and in particular it has been demonstrated that protein corresponding to 204 kDa is homologous to the beta 4 subunit of the integrin complex. It has been shown recently that monoclonal antibody to TSP-180 (MoAb 135-13C) stimulates cell growth in vitro and induces phosphorylation of the 204-kDa protein. We now report that insulin increases the phosphorylation of the 204-kDa protein 30-fold in intact carcinoma cells and epidermal growth factor (EGF) causes a threefold increase. Insulin-like growth factor (IGF-I) and platelet-derived growth factor have no effect. The effect of insulin and of IGF-I on phosphorylation of their own receptors was studied using solubilized cell membranes. Insulin and IGF-I each induced a fivefold increase in the phosphorylation of their respective receptor beta subunits. In order to test if phosphorylation of the 204-kDa protein was induced by direct binding of growth factors to TSP-180 and to identify growth factor receptors on line 1 cells, affinity cross-linking studies were performed. Affinity labeling of receptors demonstrated that insulin and IGF-I both bind to a 135-kDa protein that corresponds to the insulin and IGF-I receptor alpha subunits. Affinity labeling of EGF receptors failed to demonstrate EGF receptor molecules (175-kDa protein) on line 1 cells. Further investigations by using a different approach confirmed the very low amount of EGF receptors on line 1 cells. Direct phosphoamino acid analysis of the 204-kDa protein purified from insulin-stimulated cells demonstrated that this beta 4 integrin subunit is phosphorylated on serine and tyrosine. We conclude that beta 4 integrin molecule is a target for phosphorylation through an indirect receptor-mediated mechanism.

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Abstract: The aim of this study was to compare the metabolic effects of a high-carbohydrate (CHO), high-fiber diet with only moderate protein restriction with those of a low-CHO, low-fiber diet with a low protein content in six diabetic patients with moderate chronic renal failure. The high-CHO, high-fiber diet induced a significant improvement in blood glucose control, a significant decrease in serum cholesterol, and a significant increase in fecal nitrogen losses. Other variables evaluated were not significantly different between the two diets, except for a significant increase in serum phosphorus during the high-CHO, high-fiber diet. N balance was not significantly different from 0 at the end of either dietary period and was very similar for both diets. The high-CHO, high-fiber diet presents many beneficial metabolic effects in diabetic patients with chronic renal failure.

Abstract: The receptors for insulin and epidermal growth factor possess tyrosine-specific protein kinase activity which may play a role in mediating the biological actions of these two peptides. We have identified a 120 kDa glycoprotein (pp120) in rat liver plasma membranes which can be phosphorylated by the insulin receptor in a cell-free system and in intact cultured hepatoma cells. In the present report, we have demonstrated in a cell-free system that solubilized epidermal growth factor receptors can phosphorylate tyrosine residues in pp120.

Abstract: The present work discloses a procedure for preparation of human liver plasma membranes containing catalytically competent insulin receptor kinase. In addition to insulin promoted phosphorylation of the beta-subunit of insulin receptor kinase, insulin promoted phosphorylation of pp 120 and two other new proteins was demonstrated. The new proteins with molecular weights of 50,000 and 120,000 do not bind to WGA, pp 120 antibody or insulin receptor antibody, but bind to the antiphosphotyrosyl antibody. The identity and physiological significance of these putative substrates for insulin receptor kinase remains to be established.

Abstract: Lizard insulin receptors are evolutionarily highly conserved. Wheat germ agglutinin-purified brain membranes demonstrate the presence of an endogenous substrate (pp 105) for both the insulin and insulin-like growth factor-I receptors. Both insulin and I-insulin-like growth factor-I stimulate the phosphorylation of this endogenous substrate in a dose-dependent manner. Following insulin-stimulated autophosphorylation of the beta subunit, there is a lag period of about 5 min prior to observable phosphorylation of the endogenous substrate. Phosphoamino acid analysis of both the beta subunit as well as pp 105 reveal primarily phosphotyrosine in both the basal as well as the stimulated state.

Abstract: The insulin receptor possesses protein kinase activity, which may play a role in mediating insulin action. Recently, we have identified a glycoprotein (pp120) in rat liver plasma membranes that is phosphorylated by the solubilized insulin receptor in a cell-free system. We now report that insulin stimulates phosphorylation of pp120 in intact H-35 cells. H-35 cells were preloaded with [32P]orthophosphate to label the intracellular ATP pool. Insulin caused a 10-fold increase in the phosphorylation of its receptor and a 2-fold increase in phosphorylation of pp120 (P less than 0.001). The time course of insulin's stimulation of pp120 closely paralleled that of insulin receptor phosphorylation over the time period investigated (15-45 min). This effect had the specificity corresponding to the insulin receptor. Epidermal growth factor was inactive, and insulin-like growth factor I had approximately equal to 1% the potency of insulin in this regard. Insulin increased 32P incorporation into pp120 in a linkage that was stable to alkaline hydrolysis, as would be expected for tyrosine-specific phosphorylation. Direct phosphoamino acid analysis confirmed that insulin increased 32P incorporation into phosphotyrosine residues in pp120.

Abstract: The effect of hyperglycemia per se on glucose uptake by muscle tissue was quantitated in six controls and six type II diabetics by the forearm technique, under conditions of insulin deficiency induced by somatostatin (SRIF) infusion (0.7 mg/h). Blood glucose concentration was clamped at its basal value during the first 60 min of SRIF infusion and then raised to approximately 200 mg/dl by a variable glucose infusion. Plasma insulin levels remained at or below 5 microU/ml during SRIF infusion, including the hyperglycemic period. No appreciable difference between controls and diabetics was present in the basal state as to forearm glucose metabolism. After 60 min of SRIF infusion and euglycemia, forearm glucose uptake fell consistently from 2.1 +/- 0.7 mg X liter-1 X min-1 to 1.0 +/- 0.6 (P less than 0.05) and from 1.7 +/- .2 to 0.4 +/- 0.3 (P less than 0.02) in the control and diabetic groups, respectively. The subsequent induction of hyperglycemia caused a marked increase in both the arterial-deep venous blood glucose difference (P less than 0.02-0.01) and forearm glucose uptake (P less than 0.01-0.005). However, the response in the diabetic group was significantly greater than that observed in controls. The incremental area of forearm glucose uptake was 276 +/- 31 mg X liter-1 X 90 min and 532 +/- 81 in the control and diabetic groups, respectively (P less than 0.02). In the basal state, the forearm released lactate and alanine both in controls and diabetic subjects at comparable rates. No increment was observed after hyperglycemia, despite the elevated rates of glucose uptake. It is concluded that (1) hyperglycemia per se stimulates forearm glucose disposal to a greater extent in type II diabetics than in normal subjects; and (2) the resulting increment of glucose disposal does not accelerate the forearm release of three carbon compounds. The data support the hypothesis that hyperglycemia per se may play a compensatory role for the defective glucose disposal in type II diabetes.

Abstract: The beta-subunit of the insulin receptor possesses a tyrosine-specific protein kinase activity which may play a role in coupling insulin binding to insulin action. Previously, we have identified a substrate for the receptor-associated protein kinase in a cell-free system. This endogenous substrate (pp120), which appeared to be a glycoprotein with an apparent mol wt of 120,000, was detected in rat liver microsomes. In the present work, we have demonstrated that pp120 is localized to a highly purified preparation of rat liver plasma membranes (Neville preparation). Moreover, pp120 appears to be specific to liver, having been detected in liver from rat, monkey, and rabbit, but not in rat brain, skeletal muscle, heart, kidney, or adipocytes. As a preliminary to addressing the question of whether insulin stimulates phosphorylation of pp120 in intact cells, we have sought to identify tissue culture cell lines that contain both insulin receptors and pp120. We have succeeded in identifying pp120 in two cell lines derived from rat liver: 1) H35 hepatoma cells (Reuber hepatoma) and 2) rat hepatocytes transformed with a temperature-sensitive mutant form of SV-40 (cultivated at both permissive and nonpermissive temperatures). In conclusion, pp120 appears to be a liver-specific plasma membrane glycoprotein which serves as a substrate for phosphorylation by the insulin receptor-associated protein kinase in a soluble cell-free system. The presence of pp120 in cultured cell lines will facilitate investigation of whether the phosphorylation of pp120 in intact cells is physiologically regulated in response to insulin.

Abstract: Insulin receptors resemble receptors for certain growth factors (epidermal growth factor, platelet-derived growth factor, and insulin-like growth factor I) in that all possess tyrosine-specific protein kinase activity. These cell surface receptors resemble protein kinases encoded by viral oncogenes in that both groups of enzymes phosphorylate proteins on tyrosine. Recently, we reported that there is immunological similarity between the insulin receptor and pp60src [the protein encoded by the src oncogene of Rous sarcoma virus (RSV)]. This is supported by the observation that anti-pp60src antiserum (TBR serum) immunoprecipitated radiolabeled insulin receptors derived from cultured human cells (IM-9 lymphoblasts and U-937 monocytes) and rabbit liver. Moreover, highly purified preparations of src protein inhibit the immunoprecipitation of insulin receptors by TBR serum, and the inhibition is correlated with the src kinase activity present in the preparation used. However, two observations suggested that there were immunological differences between pp60src and mammalian insulin receptors. 1) Even at a relatively high concentration (dilution, 1:10), TBR serum immunoprecipitated a relatively small percentage (approximately 20%) of the labeled insulin receptors. 2) Some lots of TBR serum with a high titer against pp60src failed to immunoprecipitate the insulin receptor. Viral oncogenes are thought to have been derived from proto-oncogenes in the host cell. Therefore, because the chicken is the natural host for RSV, we inquired whether there might be closer homology between pp60src and avian insulin receptors. Surprisingly, under conditions where TBR serum immunoprecipitates human insulin receptors, we could not detect immunoprecipitation of avian insulin receptors from chicken liver, chicken embryo fibroblasts, or turkey erythrocytes. The immunoprecipitation of human insulin receptor is not dependent on the method used for labeling the cells ([125I]insulin cross-linking), inasmuch as the receptor labeled by autophosphorylation with [gamma-32P]ATP could also be immunoprecipitated by TBR serum. These observations suggest that there is structural homology between pp60src and the insulin receptor (most likely the beta-subunit). Nevertheless, it seems unlikely that the insulin receptor gene is the proto-oncogene for the src gene of RSV.

Abstract: The family of tyrosine-specific protein kinases includes proteins encoded by retroviral oncogenes as well as receptors for insulin and several growth factors. Antibodies to pp60src, the protein encoded by the src oncogene of Rous sarcoma virus (RSV), can specifically immunoprecipitate affinity-labeled insulin receptors from cultured human lymphocytes (IM-9 cells). This precipitation is specifically inhibited by the src gene product purified from RSV-transformed rat cells. These observations provide evidence that there is structural homology between the insulin receptors and pp60src.

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Abstract: Recent studies have demonstrated that high-carbohydrate-high-fiber diets may improve the metabolic control in diabetes. To evaluate the influence of dietary carbohydrates separate from dietary fiber on blood glucose control, six insulin-dependent diabetic patients (IDD) were assigned in random order to two weight-maintaining diets for consecutive periods of 10 days. The diets differed in carbohydrate (41% in diet A and 60% in diet B) and fat content (41% and 20%, respectively) but were identical in calories, proteins, simple sugars, and fiber. After each dietary period blood glucose was continuously monitored for 24 h (Biostator GCIIS, Life Science Instruments, Miles Laboratories, Elkhart, Indiana). The M value was 48 +/- 20 after diet A and 96 +/- 27 after diet B (t = 3.83, P less than 0.025); the mean daily blood glucose was 152 +/- 5 mg/dl after diet A and 206 +/- 11 mg/dl after diet B (t = 7.50, P less than 0.001). Similarly, the blood glucose level for the 3-h period after each of the three main meals was lower after diet A than after diet B (analysis of variance: F = 5.2, P less than 0.05). No significant difference in fasting serum cholesterol, triglycerides, or serum lipoprotein composition was observed between the two diets. In order to separate the influence of dietary carbohydrate and fat on postprandial blood glucose concentration, an additional test meal experiment was performed in eight insulin-dependent diabetic patients. In random order on consecutive days they were given two standard meals that were identical in carbohydrate and protein content and differed only in the amount of olive oil added to the meals (12 g versus 36 g).(ABSTRACT TRUNCATED AT 250 WORDS)

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Abstract: Development of diabetes mellitus is a common complication of side to side porta-caval anastomosis (PCA). Five patients with liver cirrhosis and portal hypertension have been studied with intravehous (IVGTT, 0,5 g/Kg B.W.) and oral (OGTT, 1 g/Kg B.W.) glucose tolerance tests before and three weeks after PCA. Fasting plasma glucose was 84 +/- 7 before and 87 +/- 3 mg/dl after PCA. Fasting IRI increased from 17 +/- 3 to 31 +/- 6 microU/ml. The pattern of plasma glucose and IRI response to IVGTT did not change after PCA. Plasma glucose resonse to OGTT after PCA showed only an earlier rise at 60 instead of 90 minutes, whereas IRI resonse (area under the insulin curve) was significantly enhanced (from 12.4 to 19.8 U/l, p < 0.05). These data suggest a role of gut polipeptides in determining hyperinsulinemia and insulin resistence in PCA patients.