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M Lorenzo

Publications and source records attributed to M Lorenzo.

At least 19 recordsLinked to original sources
PubMed2001

Akt mediates insulin induction of glucose uptake and up-regulation of GLUT4 gene expression in brown adipocytes.

Insulin acutely stimulated glucose uptake in rat primary brown adipocytes in a PI3-kinase-dependent but p70S6-kinase-independent manner. Since Akt represents an intermediate step between these kinases, this study investigated the contribution of Akt to insulin-induced glucose uptake by the use of a chemical compound, ML-9, as well as by transfection with a dominant-negative form of Akt (DeltaAkt). Pretreatment with ML-9 for 10 min completely inhibited insulin stimulation of (1) Akt kinase activity, (2) Akt phosphorylation on the regulatory residue Ser473 but not on Thr308, and (3) mobility shift in Akt1 and Akt2. However, ML-9 did not affect insulin-stimulated PI3-kinase nor PKCzeta activities. In consequence, ML-9 precluded insulin stimulation of glucose uptake and GLUT4 translocation to plasma membrane (determined by Western blot), without any effect on the basal glucose uptake. Moreover, DeltaAkt impaired insulin stimulation of glucose uptake and GFP-tagged GLUT4 translocation to plasma membrane in transiently transfected immortalised brown adipocytes and HeLa cells, respectively. Furthermore, ML-9 treatment for 6 h down-regulated insulin-induced GLUT4 mRNA accumulation, without affecting GLUT1 expression, in a similar fashion as LY294002. Indeed, co-transfection of brown adipocytes with DeltaAkt precluded the transactivation of GLUT4-CAT promoter by insulin in a similar fashion as a dominant-negative form of PI3-kinase. Our results indicate that activation of Akt may be an essential requirement for insulin regulation of glucose uptake and GLUT4 gene expression in brown adipocytes.

Adipocytes↗
PubMed2001

Insulin signaling leading to proliferation, survival, and membrane ruffling in C2C12 myoblasts.

We have recently shown that insulin induced myogenesis in the mouse C2C12 skeletal muscle cell line by activation of phosphatidylinositol (PI) 3-kinase/p70S6-kinase and p38-mitogen-activated protein kinase (MAPK) and downregulation of p42/p44-MAPK. This study investigated the insulin-signaling pathways involved in mitogenesis, survival, and membrane ruffling in C2C12 myoblasts, a cellular system that besides IGF-I receptors, expressed a high number of functional insulin receptors. Insulin (10 nM) rapidly stimulated beta-chain insulin receptor and IRS-1 tyrosine phosphorylation, IRS-2 being poorly and SHC not phosphorylated at all. However, an association of SHC with IRS-1 was found under insulin stimulation. Insulin stimulated IRS-1 association with p85alpha leading to the activation of PI3-kinase, and, subsequently AKT and p70S6-kinases. Moreover, both p42/p44- and p38-MAPKs resulted in phosphorylation after insulin stimulation. Insulin treatment for 24 h produced mitogenesis, as demonstrated by the increase in ((3)H)-thymidine incorporation, DNA content, the expression of PCNA and cyclin D1 proteins, and the proportion of cells in S + G2/M phases of the cell cycle. This mitogenic effect of insulin was precluded by inhibition of p70S6-kinase (either by rapamycin or by the PI3-kinase inhibitor LY294002) as well as by inhibition of p44/p42-MAPK with PD098059, but was not affected by inhibition of p38-MAPK. Serum deprivation of C2C12 myoblasts resulted in growth arrest at the GO/G1 phases of the cell cycle and apoptosis, as detected either by DNA laddering or by increase in the percentage of hypodiploid cells. Insulin rescued serum-deprived cells from apoptosis in an AKT-dependent manner, as demonstrated by the inhibition of AKT-activity by the use of LY294002 and ML-9, meanwhile neither inhibition of p70S6-kinase, nor MAPK affected insulin-induced survival. Finally, we evaluated the capacity of insulin to modulate actin cytoskeleton rearrangement. Insulin stimulation of myoblasts produced membrane ruffling and decreased actin stress fibers; this biological response being dependent of p38-MAPK, as demonstrated by the use of the p38-MAPK inhibitors SB203580 or PD169316, but independent of PI3-kinase and p42/p44-MAPK.

Adaptor Proteins, Signal Transducing↗
PubMed2001

Insulin produces myogenesis in C2C12 myoblasts by induction of NF-kappaB and downregulation of AP-1 activities.

In the present study, we have examined the insulin-signaling pathways involved in myogenesis in mouse C2C12 skeletal muscle cell line, a cellular system that expresses high number of high affinity insulin receptors. Insulin (50 nM) rapidly (5 min) stimulated beta-chain insulin receptor, activated the phosphatidylinositol (PI) 3-kinase/Akt/p70S6-kinase signaling pathway, as well as phosphorylated both p44/p42- and p38-mitogen-activated protein kinases (MAPKs). Preconfluent cells were differentiated in a serum-free medium in response to 50 nM insulin for 72 h, as revealed by the formation of multinucleated myotubes and the induction of the creatine kinase activity. This differentiation process was also monitored by the inhibition of the PCNA content and induction of the cell cycle inhibitor p21. Furthermore, insulin induced nuclear factor-kappaB (NF-kappaB) DNA binding activity and down-regulated activating protein-1 (AP-1) DNA binding activity throughout the differentiation process. The use of specific inhibitors of the insulin-signaling pathways indicated that myogenesis was precluded by treatment for 72 h with LY294002 (an inhibitor of PI 3-kinase), rapamycin (a p70S6-kinase blocker), and SB203580 or PD169316 (p38-MAPK inhibitors). These inhibitors abolished insulin induction of NF-kappaB DNA binding activity and kappaB-chloramphenicol acetyltransferase (CAT) promoter activity, maintaining expressed cytosolic IkappaB-alpha protein, and increased AP-1 DNA binding activity and TRE-CAT promoter activity. These data suggest that insulin induces myogenesis in C2C12 through PI 3-kinase/ p70S6-kinase and p38-MAPK pathways, the signaling through p44/p42-MAPK being inhibited.

Animals↗
PubMed2001

Traumatic intrapericardial diaphragmatic hernia: case report and literature review.

A traumatic rupture of the diaphragm, with herniation of the abdominal viscera into the thoracic cavity, is relatively rare, being observed in approximately 3-7% of all abdominal or thoracic traumas [1]. Herniation into the pericardium is very rare [2]. We have reviewed the post-traumatic intrapericardial diaphragmatic hernias (TIPDH) published in the world literature within our reach and, as it was already stated in the review carried out by Van Loenhout et al. [3] in 1986, approximately one post-traumatic intrapericardial hernia is reported every year. The patient that we present makes case 82 of those published until October of 1999.

Accidents, Traffic↗
PubMed2001

Conversion of Lap-Band to gastric bypass for dilated gastric pouch.

An 18-year-old female who had undergone a laparoscopic adjustable gastic banding developed several episodes of gastric pouch dilatation (GPD), treated conservatively. The last GPD (31 months after Lap-Band placement) involved the lesser curvature of the stomach and was refractory to medical treatment. Conversion to an open gastric bypass was performed. Gastric bypass is an option in the case of Lap-Band failure.

Adolescent↗
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↗
PubMed2000

Akt mediates insulin rescue from apoptosis in brown adipocytes: effect of ceramide.

We have recently shown that insulin can rescue serum deprived adipocytes from apoptosis in a PI 3 kinase and MAP kinase dependent manner. This study investigated the contribution of Akt and p70S6-kinase in insulin rescue from two different apoptotic triggers, serum deprivation and ceramide treatment. Insulin rescued serum-deprived immortalized brown adipocytes from apoptosis through phosphatidylinositol (PI) 3-kinase and Akt pathways, but independently of p70S6-kinase, as demonstrated by the use of inhibitors such as LY294002 or Rapamycin, and transfection experiments with dominant-negative constructs of Akt or p85 subunit of PI 3-kinase. A constitutively active Akt construct mimicked the insulin survival effect, decreasing the percentage of hypodiploid cells, the percentage of apoptopic cells and precluding the formation of apoptotic nuclei. We propose that the insulin survival effect on immortalized brown adipocytes is mediated through activation of Akt. However, insulin and EGF failed to rescue brown adipocytes from ceramide-induced apoptosis, as determined by DNA laddering, hypodiploid cells and apoptotic nuclei. Ceramide treatment blunted Akt activity but not PI 3-kinase activity, and insulin and EGF were unable to activate Akt. Ceramide also caused apoptosis in cells transfected with a constitutively active Akt construct, since phosphorylation of Akt was impaired under these experimental conditions. This study suggests that activation of Akt may be an absolute requirement for the survival of brown adipocytes.

Adipocytes↗
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↗