PubMed Health⌕ Search

Biomedical subjects

M Lorenzo

Publications and source records attributed to M Lorenzo.

At least 19 recordsLinked to original sources
PubMed2000

Okadaic acid inhibits insulin-induced glucose transport in fetal brown adipocytes in an Akt-independent and protein kinase C zeta-dependent manner.

In the present study we have investigated the effect of increased serine/threonine phosphorylation of insulin receptor substrates-1 and -2 (IRS-1 and IRS-2) by okadaic acid pretreatment on brown adipocyte insulin signalling leading to glucose transport, an important metabolic effect of insulin in brown adipose tissue. Okadaic acid pretreatment before insulin stimulation decreased IRS-1 and IRS-2 tyrosine phosphorylation in parallel to a decrease in their sodium dodecyl sulfate-polyacrylamide gel electrophoresis mobility. IRS-1/IRS-2-associated p85alpha and phosphatidylinositol (PI) 3-kinase enzymatic activity were partly reduced in brown adipocytes pretreated with okadaic acid upon stimulation with insulin. Furthermore, insulin-induced glucose uptake was totally abolished by the inhibitor in parallel with a total inhibition of insulin-induced protein kinase C (PKC) zeta activity. However, activation of Akt/PKB or p70 S6 kinase (p70(s6k)) by insulin remained unaltered. Our results suggest that downstream of PI 3-kinase, insulin signalling diverges into at least two independent pathways through Akt/PKB and PKC zeta, the PKC zeta pathway contributing to glucose transport induced by insulin in fetal brown adipocytes.

Adipose Tissue, Brown↗
PubMed1999

Activated Ha-ras induces apoptosis by association with phosphorylated Bcl-2 in a mitogen-activated protein kinase-independent manner.

Serum deprivation of Ha-ras-transformed brown adipocyte cell line resulted in a dramatic apoptotic cell death, as detected either by DNA laddering or by an increase in the percentage of hypodiploid cells or by nuclei condensation and fragmentation, as compared with immortalized cell line or primary fetal brown adipocytes. Moreover, transient transfection of immortalized brown adipocytes with a constitutively active ras gene (Ha-raslys12) mimics the high rate of apoptosis detected in the transformed cell line. On the other hand, transient transfection of the dominant-negative construct of raf-1 rescued serum-deprived Ha-ras-transformed brown adipocytes from apoptosis, decreasing the percentage of hypodiploid cells, the external display of phosphatidylserine, and the DNA laddering. However, inhibition of mitogen-activated protein kinase with PD098059 did not preclude apoptosis and in fact increased the rate of apoptosis observed in serum-deprived Ha-ras-transformed cells, indicating that the Ras/Raf-1 pathway induced apoptosis throughout a mitogen-activated protein kinase kinase 1 (MEK-1)-independent pathway. Furthermore, apoptosis in Ha-ras-transformed brown adipocytes is concurrent with an up-regulation in the expression of the pro-apoptotic protein Bcl-xS, the expression of the anti-apoptotic protein Bcl-2 being down-regulated. Finally, an association of Ras and Raf with phosphorylated Bcl-2 protein was demonstrated in immunoprecipitates from apoptotic cells. Thus, we propose a mechanism of apoptosis in Ha-ras-transformed adipocytes under serum deprivation involving Raf-1 association with phosphorylated Bcl-2, down-regulation of Bcl-2 expression, and up-regulation of Bcl-xS expression.

Adipocytes↗
PubMed1999

Inhibition of caspases rescues brown adipocytes from apoptosis downregulating BCL-XS and upregulating BCL-2 gene expression.

Serum deprivation of the immortalized brown adipocyte cell line resulted in growth arrest in G0/G1 phases of the cell cycle and apoptosis, as detected by DNA laddering, nuclei condensation and fragmentation, and an increase in the percentage of hypodiploid cells. In addition, apoptosis in these cells is accompanied by an induction of the expression of the apoptotic form of the Bcl-x gene, the isoform Bcl-xS, and by a decrease of Bcl-2 expression, Bcl-xL remaining almost undetectable. The loss of mitochondrial membrane potential was associated with apoptosis. Z-VAD, a cell-permeable inhibitor of caspases, but not cycloheximide, precludes DNA laddering under serum deprivation. Moreover, Z-VAD rescues serum-deprived brown adipocytes from apoptosis, decreasing the percentage of hypodiploid cells, the percentage of apoptotic cells under Tunnel assay, and the external display of phosphatidylserine. More importantly, Z-VAD survival effects on immortalized brown adipocytes concur with a downregulation of Bcl-xS mRNA/protein and an upregulation of Bcl-2 protein content. Ultimately, Z-VAD prevents the loss of mitochondrial membrane potential.

Adipocytes↗
PubMed1999

Insulin and insulin-like growth factor I up-regulate GLUT4 gene expression in fetal brown adipocytes, in a phosphoinositide 3-kinase-dependent manner.

Fetal brown adipocytes cultured in a serum-free medium, containing 5 mM glucose, expressed both GLUT4 and GLUT1 glucose transporters at the mRNA and protein level. Treatment with either insulin or insulin-like growth factor (IGF)-I at physiological concentrations up-regulates the expression of the GLUT4 gene, producing a time-dependent mRNA accumulation (7-fold increase at 24 h) and a 2.5-fold increase in the amount of protein in the total membrane fraction. However, insulin treatment down-regulates GLUT1 mRNA and protein expression. Moreover, either insulin or IGF-I transactivates a full-promoter GLUT4-chloramphenicol acetyltransferase gene (CAT) construct transiently transfected to the cells, without affecting GLUT1-CAT activity. In consequence, insulin treatment for 24 h increased by 3-fold the basal glucose uptake. Inhibition of phosphoinositide (PI) 3-kinase activity with chemical agents such as wortmannin or LY294002 partially blocked insulin-induced GLUT4 mRNA accumulation, insulin-induced GLUT4 protein content, GLUT4-CAT transactivation and glucose uptake. Furthermore, co-transfection of brown adipocytes with a dominant-negative form of PI 3-kinase precluded the transactivation of the GLUT4 promoter by insulin. However, inhibition of p70S6 kinase (p70(s6k)) with rapamycin or of mitogen-activated protein kinase (MAPK) with PD098059 does not preclude insulin effects on GLUT4 gene expression or glucose uptake. Our results show for the first time a positive effect of insulin on GLUT4 gene expression in fetal brown adipocytes, suggesting the existence of insulin response element(s) in its promoter. Moreover, PI 3-kinase, but not p70(s6k) or MAPK, is an essential requirement for insulin regulation of GLUT4 gene expression.

Adipose Tissue↗
PubMed1999

Differences between men and women as regards the effects of protein-energy malnutrition on the hypothalamic-pituitary-gonadal axis.

Although protein-energy malnutrition (PEM) affects 50% of hospitalized patients, its effects on the hypothalamic-pituitary-gonadal (HPG) axis have not been extensively investigated. To investigate the effects of PEM on the HPG axis in hospitalized patients, 62 inpatients ages 18-91 y (35 men and 27 women) had a nutritional and hormonal evaluation. Hormones were determined in blood samples obtained between 0700 and 1200 h. Patients were divided into two subgroups: those with body mass index (BMI) <18.5 kg/m2 (low body mass index [LBMI]; 16 men, 13 women) and those with BMI >20 kg/m2 (normal-high body mass index [NHBMI]; 19 men, 14 women). The nutritional parameters of LBMI patients were inferior to those of NHBMI patients. Total and free testosterone levels were subnormal, 31.4% and 17.2% respectively, in all men; free testosterone was subnormal in 31.25% of LBMI versus 5.3% of NHBMI male patients and total testosterone concentration was subnormal in 43.8% of LBMI versus 21.1% of NHBMI male patients. Luteinizing hormone (LH) level was higher in LBMI men than in NHBMI men, whereas the reverse was the case for women, for whom follicle-stimulating hormone (FSH) also was lower in the LBMI group than the NHBMI group. The HPG axis hormones which best discriminated between the LBMI and NHBMI groups were free testosterone for men and LH and FSH for women, which were all lower in the LBMI than in the NHBMI group. LH was correlated with BMI and midupper arm muscle circumference (AMC) (women positively and men negatively) but not with triceps skin-fold thickness (TSF). Total testosterone level was positively correlated with AMC and free testosterone with TSF. Hypogonadism is common among hospitalized patients with PEM. Men with PEM have low testosterone levels with normal or high gonadotropin levels, which suggests impairment of Leydig cell function. Women with PEM suffer hypogonadotropic hypogonadism. AMC correlates positively with total serum testosterone concentration in men and with LH levels in women, suggesting that satisfactory function of the HPG axis requires a functional (protein) reserve as well as an energy (fat) reserve.

Adult↗
PubMed1999

Treatment of morbid obesity and gastroesophageal reflux with hiatal hernia by Lap-Band.

BACKGROUND: Esophageal reflux is common in obese patients. Hiatal hernia is considered a potential contraindication to placement of a Lap-Band. METHODS: Esophageal investigation in patients who were candidates for a Lap-Band included clinical evaluation of symptoms (scoring system), endoscopic and radiologic evaluation, 24-h pH test, and stationary manometry. Patients with gastroesophageal reflux (GER) with or without hiatal hernia underwent the Lap-Band procedure. RESULTS: GER was diagnosed in 12/40 morbidly obese patients, 11 of whom received a standard Lap-Band (3 patients were radiologically diagnosed with transient hiatal hernia). One patient with a large hiatal hernia underwent closure of the diaphragmatic esophageal hiatus, and the Lap-Band was positioned similarly to an Angelchik prosthesis. All but 1 patient who was lost at follow-up were symptom-free (range 1-24 months). CONCLUSION: GER with or without hiatal hernia is not a contraindication for obese patients undergoing a Lap-Band procedure. It accomplishes by a single operation satisfactory treatment of these two disturbing diseases.

Adult↗
PubMed1999

Follow-up of Lap-Band complications.

BACKGROUND: Proximal gastric pouch dilation (PGPD) and band dislocation (BD) are the most frequent complications of laparoscopic adjustable silicone gastric banding (LASGB). METHODS: Conservative treatment of PGPD and BD was attempted in all patients by deflation of the band. In the case of failure, laparoscopic exploration was performed. RESULTS: From January 1996 to July 1998, 8 of 40 patients who underwent LASGB experienced PGPD (n = 7) or BD (n = 1). Debanding was performed in 3 patients with PGPD, while in 4 the pouch dilation was successfully treated with deflation of the band. Two patients (PGPD and BD) were treated with band repositioning. Weight loss was not influenced in patients treated conservatively, compared with patients who did not experience complications. CONCLUSIONS: PGPD and BD are not always responsible for band failure in LASGB. Conservative treatment can be successful, and repositioning of the band is feasible in selected cases.

Adult↗
PubMed1998

H-ras induces glucose uptake in brown adipocytes in an insulin- and phosphatidylinositol 3-kinase-independent manner.

Fetal brown adipocytes (parental cells) expressed mainly Glut4 mRNA glucose transporter, the expression of Glut1 mRNA being much lower. At physiological doses, insulin stimulation for 15 min increased 3-fold glucose uptake and doubled the amount of Glut4 protein located at the plasma membrane. Moreover, phosphatidylinositol (PI) 3-kinase activity was induced by the presence of insulin in those cells, glucose uptake being precluded by PI 3-kinase inhibitors such as wortmannin or LY294002. H-raslys12-transformed brown adipocytes showed a 10-fold higher expression of Glut1 mRNA and protein than parental cells, Glut4 gene expression being completely down-regulated. Glucose uptake increased by 10-fold in transformed cells compared to parental cells; this uptake was unaltered in the presence of insulin and/or wortmannin. Transient transfection of parental cells with a dominant form of active Ras increased basal glucose uptake by 5-fold, no further effects being observed in the presence of insulin. However, PI 3-kinase activity (immunoprecipitated with anti-alphap85 subunit of PI 3-kinase) remained unaltered in H-ras permanent and transient transfectants. Our results indicate that activated Ras induces brown adipocyte glucose transport in an insulin-independent manner, this induction not involving PI 3-kinase activation.

Adipocytes↗
PubMed1998

Insulin/IGF-I rescues immortalized brown adipocytes from apoptosis down-regulating Bcl-xS expression, in a PI 3-kinase- and map kinase-dependent manner.

Serum deprivation of immortalized brown adipocyte cell line resulted in growth arrest in G0/G1 phases of the cell cycle and apoptosis, as detected either by DNA laddering or by increase in the percentage of hypodiploid cells. Furthermore, apoptosis is concurrent with a dramatic increase in the expression of the proapoptotic protein Bcl-xS, the expression of Bcl-xL remaining almost undetectable. Insulin/insulin-like growth factor (IGF-I) rescued serum-deprived brown adipocytes from apoptosis, decreasing the number of hypodiploid cells and increasing the number of cells undergoing cell cycle progression throughout S and G2/M phases of the cell cycle. Moreover, insulin down-regulated Bcl-xS expression without inducing the expression of Bcl-xL. Both phosphatidylinositol (PI) 3-kinase and mitogen-activated protein kinase (MAPK) pathways are necessary for insulin/IGF-I full survival effect, since the use of specific inhibitors of PI 3-kinase activity (wortmannin or LY294002, at the dose that inhibits PI 3-kinase activity induced by insulin) or MAPK kinase activity inhibitor (PD098059, at the dose that inhibits insulin-induced phosphorylation of MAPK) totally blocked the antiapoptotic effect induced by insulin/IGF-I, respectively. In conclusion, insulin survival effect on immortalized brown adipocytes is associated with inhibition of the Bcl-xS content without changing Bcl-xL, in a PI 3-kinase- and MAP kinase-dependent manner.

Adipocytes↗
PubMed1998

Inhibition of PI 3-kinase and RAS blocks IGF-I and insulin-induced uncoupling protein 1 gene expression in brown adipocytes.

Fetal brown adipocytes expressed uncoupling protein 1 (UCP1) mRNA, this expression being blunted throughout culture for 24 h in a serum-free medium. At physiological doses, either insulin-like growth factor I (IGF-I) or insulin turned out to be as potent as dibutyryl cAMP (dbcAMP) in increasing UCP1 gene transcription rate (1 h) and also UCP1 mRNA accumulation (3 h), their maximal effect (15-fold increase) reached upon treatment for 24 h. Upon treatment with either IGF-I or insulin for 48 h, a 7-fold increase in the UCP1 protein content relative to levels in the control cells was found, this induction being abolished in the presence of cycloheximide. Moreover, either IGF-I or insulin transactivates the UCP1-chloramphenicol acetyl transferase (CAT) fusion gene after transient transfection of primary brown adipocytes, these effects being tissue-specific. Transient transfection of dominant-negative form of phosphatidylinositol (PI) 3-kinase completely blocked the transactivation of the fusion gene UCP1-CAT induced by either IGF-I or insulin, although inhibition of p70S6kinase with rapamycin does not preclude transactivation of the UCP1 promoter by insulin. Furthermore, transient transfection of dominant-negative form of p21-ras or treatment of cells with a mitogen-activated protein kinase kinase (MEK-1) inhibitor (PD098059) completely abolished insulin-induced UCP1-CAT transactivation. Cotransfection with dominant-negative p85 or with dominant-negative Ras also produced down-regulation of the insulin or IGF-I-induced 12-O-tetradecanoylphorbol-13-acetate response element (TRE)-CAT (five AP-1, activating protein-1, binding sites arranged in tandem) transactivation. In addition, insulin induced AP-1 DNA binding activity, this effect being totally prevented in the presence of MEK-1 inhibitor. These results strongly suggest that either IGF-I or insulin induced thermogenic-differentiation through AP-1 activity in a PI 3-kinase and Ras/MAPK dependent manner in brown adipocytes.

Adipocytes↗
PubMed1998

Can Ringer's lactate be used safely with blood transfusions?

BACKGROUND: Blood bank recommendations specify that Ringer's lactate solution (LR) should be avoided while transfusing blood. However, there are few studies either evaluating or quantifying increased coagulation during rapid infusion of LR and blood. DESIGN AND METHODS: Whole blood (WB, n = 25) and packed red blood cells (PRBC, n = 26) were rapidly admixed with normal saline (NS), Lactate solution and LR with 1 g (LR-1), 2 g (LR-2), and 5 g (LR-5) CaCl2/L solutions for assessment of infusion time, filter weight, and clot formation. RESULTS: No significant differences in infusion time or filter weight using WB or PRBC with NS or LR were seen. No significant difference in clot formation between NS and LR with WB or PRBC was found, but the presence of visible clot was increased in the LR-5 group (P = 0.013, WB, and P = 0.002, PRBC). CONCLUSION: A comparison of LR and NS with rapid infusion rates of blood showed no significant difference between infusion time, filter weight and clot formation. Blood bank guidelines should be revised to allow the use of LR in the rapid transfusion of PRBC.

Blood Coagulation↗
PubMed1998

Tumor necrosis factor-alpha causes insulin receptor substrate-2-mediated insulin resistance and inhibits insulin-induced adipogenesis in fetal brown adipocytes.

Treatment of fetal brown adipocytes with 0.6 nM tumor necrosis factor (TNF)-alpha for 24 h resulted in a partial impairment in the expression of fatty acid synthase, glycerol-3-phosphate dehydrogenase, and glucose transporter (GLUT)-4 messenger RNAs (mRNAs), as well as in the enhancement in the cytoplasmic lipid content in response to insulin. However, the expression of the tissue-specific gene, uncoupling protein 1, is increased by the presence of TNF-alpha. The antiadipogenic effect of TNF-alpha was accompanied by a down-regulation of CCAAT/enhancer-binding protein-alpha and beta mRNAs and up-regulation of CCAAT/enhancer-binding protein-delta, with the expression of peroxisome proliferator-activated receptor-gamma remaining essentially unmodified. Moreover, TNF-alpha caused an insulin resistance on the insulin-induced glucose uptake in brown adipocytes. Pretreatment with TNF-alpha resulted in hypophosphorylation of the insulin receptor in response to insulin, without affecting the number of insulin receptors per cell or its molecular mass. However, insulin receptor substrate (IRS)-1 and IRS-2 signaling in response to insulin showed functional differences. Thus, TNF-alpha pretreatment induced a hypophosphorylation of IRS-2 but not of IRS-1. This effect leads to an impairment in the IRS-2-associated phosphatidylinositol (PI) 3-kinase activation due to a decreased association of alpha-p85 regulatory subunit of PI 3-kinase with IRS-2 but not in the IRS-1-associated PI 3-kinase activation in response to insulin. Our results indicate that TNF-alpha induced an IRS-2- but not IRS-1-mediated insulin resistance on glucose transport and lipid synthesis in fetal brown adipocytes.

Adipocytes↗
PubMed1998

Insulin receptor substrate (IRS) proteins IRS-1 and IRS-2 differential signaling in the insulin/insulin-like growth factor-I pathways in fetal brown adipocytes.

In the present study we have investigated the contribution of the insulin receptor substrate proteins (IRS-1 and IRS-2) to the insulin/insulin like growth factor I (IGF-I)-signaling pathways in fetal rat brown adipocytes, a model that expresses both insulin and IGF-I receptors. Insulin/IGF-I rapidly stimulated IRS-1 and IRS-2 tyrosine phosphorylation, their association with p85alpha, and IRS-1- and IRS-2-associated phosphatidylinositol (PI) 3-kinase activation to the same extent, the effect of insulin being stronger than the effect of IGF-I at the same physiological dose (10 nM). Furthermore, insulin/IGF-I stimulated IRS-1-associated Grb-2 phosphorylation. However, IRS-2-associated Grb-2 phosphorylation was barely detected. Pull-down experiments with glutathione-S-transferase-fusion proteins containing SH2-domains of p85alpha revealed a strong association between IRS-1 and IRS-2 with p85alpha in response to insulin/IGF-I, the insulin effect being stronger than IGF-I. However, the Grb-2-SH2 domain showed functional differences. While a strong association between IRS-1/Grb-2 was found, IRS-2/Grb-2 association was virtually absent in response to insulin/IGF-I, as also demonstrated in competition studies with a phosphopeptide containing the phosphotyrosine 895 residue within the putative Grb-2-binding domain. Finally, insulin/IGF-I stimulated tyrosine phosphorylation of the three SHC proteins (46, 52, and 66 kDa). Moreover, insulin/IGF-I markedly increased the amount of Grb-2-associated SHC proteins by the same extent. Our results suggest that both IRS-1 and IRS-2 are required for phosphatidylinositol 3-kinase activation that leads to adipogenic and thermogenic differentiation of fetal brown adipose tissue; meanwhile, IRS-1 and SHC, but not IRS-2, associate with Grb-2 leading to the ras-mitogen-activated protein kinase-signaling pathway required for fetal brown adipocyte proliferation.

Adaptor Proteins, Signal Transducing↗
PubMed1997

Spinal cord injury. A preventable public burden.

BACKGROUND: Spinal cord injury (SCI) is a devastating occurrence with important consequences for the individual and society. Previous studies have documented the epidemiology and costs of SCI and the rehabilitation needs after SCI; however, data about the preventability of SCI are lacking. OBJECTIVE: To test the hypotheses that most SCIs are preventable and that much of the cost of SCI is borne by the public. DESIGN: Retrospective review of medical records and trauma registry data. SETTING: A 417-bed county hospital with a level I trauma center. METHODS: To evaluate the preventability of SCI, the records of trauma patients sustaining SCI from July 1, 1990, through June 30, 1995, were reviewed. The criteria for preventability of blunt injuries included the following: failure to use restraint devices; intoxication of drivers, motorcyclists, or pedestrians; and falls or diving accidents involving the use of drugs or alcohol. The criteria for preventability of penetrating injuries included the following: illegal possession of a firearm, accidental discharge of a weapon, and suicide attempts. Statistics were performed with the paired Student t test and chi 2 with significance attributed to a P value less than .05. RESULTS: Spinal cord injury occurred in 150 patients; 71% of the injuries were the result of blunt trauma. Injury was potentially preventable in 74% of the blunt injuries and 66% of the penetrating injuries (P = .15). Patients with a penetrating SCI were younger (P < .001) and relied more on public funding than did those with a blunt SCI (65% vs 81%; P = .05). CONCLUSIONS: Most SCIs are preventable with strict enforcement of existing statutes. Furthermore, the financial burden of these preventable injuries is largely borne by the public.

Critical Care↗