Single administration of recombinant IL-6 restores the gene expression of lipogenic enzymes in liver of fasting IL-6-deficient mice.

BACKGROUND AND PURPOSE: Lipogenesis is intimately controlled by hormones and cytokines as well as nutritional conditions. IL-6 participates in the regulation of fatty acid metabolism in the liver. We investigated the role of IL-6 in mediating fasting/re-feeding changes in the expression of hepatic lipogenic enzymes. EXPERIMENTAL APPROACH: Gene and protein expression of lipogenic enzymes were examined in livers of wild-type (WT) and IL-6-deficient (IL-6(-/-) ) mice during fasting and re-feeding conditions. Effects of exogenous IL-6 administration on gene expression of these enzymes were evaluated in vivo. The involvement of STAT3 in mediating these IL-6 responses was investigated by using siRNA in human HepG2 cells. KEY RESULTS: During feeding, the up-regulation in the hepatic expression of lipogenic genes presented similar time kinetics in WT and IL-6(-/-) mice. During fasting, expression of lipogenic genes decreased gradually over time in both strains, although the initial drop was more marked in IL-6(-/-) mice. Protein levels of hepatic lipogenic enzymes were lower in IL-6(-/-) than in WT mice at the end of the fasting period. In WT, circulating IL-6 levels paralleled gene expression of hepatic lipogenic enzymes. IL-6 administration in vivo and in vitro showed that IL-6-mediated signalling was associated with the up-regulation of hepatic lipogenic enzyme genes. Moreover, silencing STAT3 in HepG2 cells attenuated IL-6 mediated up-regulation of lipogenic gene transcription levels. CONCLUSIONS AND IMPLICATIONS: IL-6 sustains levels of hepatic lipogenic enzymes during fasting through activation of STAT3. Our findings indicate that clinical use of STAT3-associated signalling cytokines, particularly against steatosis, should be undertaken with caution.

Impaired autophagic flux is associated with increased endoplasmic reticulum stress during the development of NAFLD.

The pathogenic mechanisms underlying the progression of non-alcoholic fatty liver disease (NAFLD) are not fully understood. In this study, we aimed to assess the relationship between endoplasmic reticulum (ER) stress and autophagy in human and mouse hepatocytes during NAFLD. ER stress and autophagy markers were analyzed in livers from patients with biopsy-proven non-alcoholic steatosis (NAS) or non-alcoholic steatohepatitis (NASH) compared with livers from subjects with histologically normal liver, in livers from mice fed with chow diet (CHD) compared with mice fed with high fat diet (HFD) or methionine-choline-deficient (MCD) diet and in primary and Huh7 human hepatocytes loaded with palmitic acid (PA). In NASH patients, significant increases in hepatic messenger RNA levels of markers of ER stress (activating transcription factor 4 (ATF4), glucose-regulated protein 78 (GRP78) and C/EBP homologous protein (CHOP)) and autophagy (BCN1) were found compared with NAS patients. Likewise, protein levels of GRP78, CHOP and p62/SQSTM1 (p62) autophagic substrate were significantly elevated in NASH compared with NAS patients. In livers from mice fed with HFD or MCD, ER stress-mediated signaling was parallel to the blockade of the autophagic flux assessed by increases in p62, microtubule-associated protein 2 light chain 3 (LC3-II)/LC3-I ratio and accumulation of autophagosomes compared with CHD fed mice. In Huh7 hepatic cells, treatment with PA for 8 h triggered activation of both unfolding protein response and the autophagic flux. Conversely, prolonged treatment with PA (24 h) induced ER stress and cell death together with a blockade of the autophagic flux. Under these conditions, cotreatment with rapamycin or CHOP silencing ameliorated these effects and decreased apoptosis. Our results demonstrated that the autophagic flux is impaired in the liver from both NAFLD patients and murine models of NAFLD, as well as in lipid-overloaded human hepatocytes, and it could be due to elevated ER stress leading to apoptosis. Consequently, therapies aimed to restore the autophagic flux might attenuate or prevent the progression of NAFLD.

Pivotal role of protein tyrosine phosphatase 1B (PTP1B) in the macrophage response to pro-inflammatory and anti-inflammatory challenge.

Inhibition of protein tyrosine phosphatase 1B (PTP1B) has been suggested as an attractive target to improve insulin sensitivity in different cell types. In the present work, we have investigated the effect of PTP1B deficiency on the response of human and murine macrophages. Using in vitro and in vivo approaches in mice and silencing PTP1B in human macrophages with specific siRNAs, we have demonstrated that PTP1B deficiency increases the effects of pro-inflammatory stimuli in both human and rodent macrophages at the time that decreases the response to alternative stimulation. Moreover, the absence of PTP1B induces a loss of viability in resting macrophages and mainly after activation through the classic pathway. Analysis of early gene expression in macrophages treated with pro-inflammatory stimuli confirmed this exacerbated inflammatory response in PTP1B-deficient macrophages. Microarray analysis in samples from wild-type and PTP1B-deficient macrophages obtained after 24 h of pro-inflammatory stimulation showed an activation of the p53 pathway, including the excision base repair pathway and the insulin signaling pathway in the absence of PTP1B. In animal models of lipopolysaccharide (LPS) and D-galactosamine challenge as a way to reveal in vivo inflammatory responses, animals lacking PTP1B exhibited a higher rate of death. Moreover, these animals showed an enhanced response to irradiation, in agreement with the data obtained in the microarray analysis. In summary, these results indicate that, although inhibition of PTP1B has potential benefits for the treatment of diabetes, it accentuates pro-inflammatory responses compromising at least macrophage viability.

Insulin directly stimulates VEGF-A production in the glomerular podocyte.

Podocytes are critically important for maintaining the integrity of the glomerular filtration barrier and preventing albuminuria. Recently, it has become clear that to achieve this, they need to be insulin sensitive and produce an optimal amount of VEGF-A. In other tissues, insulin has been shown to regulate VEGF-A release, but this has not been previously examined in the podocyte. Using in vitro and in vivo approaches, in the present study, we now show that insulin regulates VEGF-A in the podocyte in both mice and humans via the insulin receptor (IR). Insulin directly increased VEGF-A mRNA levels and protein production in conditionally immortalized wild-type human and murine podocytes. Furthermore, when podocytes were rendered insulin resistant in vitro (using stable short hairpin RNA knockdown of the IR) or in vivo (using transgenic podocyte-specific IR knockout mice), podocyte VEGF-A production was impaired. Importantly, in vivo, this occurs before the development of any podocyte damage due to podocyte insulin resistance. Modulation of VEGF-A by insulin in the podocyte may be another important factor in the development of glomerular disease associated with conditions in which insulin signaling to the podocyte is deranged.

Protein tyrosine phosphatase 1B modulates GSK3ß/Nrf2 and IGFIR signaling pathways in acetaminophen-induced hepatotoxicity.

Acute hepatic failure secondary to acetaminophen (APAP) poisoning is associated with high mortality. Protein tyrosine phosphatase 1B (PTP1B) is a negative regulator of tyrosine kinase growth factor signaling. In the liver, this pathway confers protection against injury. However, the involvement of PTP1B in the intracellular networks activated by APAP is unknown. We have assessed PTP1B expression in APAP-induced liver failure in humans and its role in the molecular mechanisms that regulate the balance between cell death and survival in human and mouse hepatocytes, as well as in a mouse model of APAP-induced hepatotoxicity. PTP1B expression was increased in human liver tissue removed during liver transplant from patients for APAP overdose. PTP1B was upregulated by APAP in primary human and mouse hepatocytes together with the activation of c-jun (NH2) terminal kinase (JNK) and p38 mitogen-activated protein kinase (p38 MAPK), resulting in cell death. Conversely, Akt phosphorylation and the antiapoptotic Bcl2 family members BclxL and Mcl1 were decreased. PTP1B deficiency in mouse protects hepatocytes against APAP-induced cell death, preventing glutathione depletion, reactive oxygen species (ROS) generation and activation of JNK and p38 MAPK. APAP-treated PTP1B(-/-) hepatocytes showed enhanced antioxidant defense through the glycogen synthase kinase 3 (GSK3)ß/Src kinase family (SKF) axis, delaying tyrosine phosphorylation of the transcription factor nuclear factor-erythroid 2-related factor (Nrf2) and its nuclear exclusion, ubiquitination and degradation. Insulin-like growth factor-I receptor-mediated signaling decreased in APAP-treated wild-type hepatocytes, but was maintained in PTP1B(-/-) cells or in wild-type hepatocytes with reduced PTP1B levels by RNA interference. Likewise, both signaling cascades were modulated in mice, resulting in less severe APAP hepatotoxicity in PTP1B(-/-) mice. Our results demonstrated that PTP1B is a central player of the mechanisms triggered by APAP in hepatotoxicity, suggesting a novel therapeutic target against APAP-induced liver failure.

Differential mitogenic signaling in insulin receptor-deficient fetal pancreatic beta-cells.

Insulin receptor (IR) may play an essential role in the development of beta-cell mass in the mouse pancreas. To further define the function of this signaling system in beta-cell development, we generated IR-deficient beta-cell lines. Fetal pancreata were dissected from mice harboring a floxed allele of the insulin receptor (IRLoxP) and used to isolate islets. These islets were infected with a retrovirus to express simian virus 40 large T antigen, a strategy for establishing beta-cell lines (beta-IRLoxP). Subsequently, these cells were infected with adenovirus encoding cre recombinase to delete insulin receptor (beta-IR(-/-)). beta-Cells expressed insulin and Pdx-1 mRNA in response to glucose. In beta-IRLoxP beta-cells, p44/p42 MAPK and phosphatidylinositol 3 kinase pathways, mammalian target of rapamycin (mTOR), and p70S(6)K phosphorylation and beta-cell proliferation were stimulated in response to insulin. Wortmannin or PD98059 had no effect on insulin-mediated mTOR/p70S(6)K signaling and the corresponding mitogenic response. However, the presence of both inhibitors totally impaired these signaling pathways and mitogenesis in response to insulin. Rapamycin completely blocked insulin-activated mTOR/p70S(6)K signaling and mitogenesis. Interestingly, in beta-IR(-/-) beta-cells, glucose failed to stimulate phosphatidylinositol 3 kinase activity but induced p44/p42 MAPKs and mTOR/p70S(6)K phosphorylation and beta-cell mitogenesis. PD98059, but not wortmannin, inhibited glucose-induced mTOR/p70S(6)K signaling and mitogenesis in those cells. Finally, rapamycin blocked glucose-mediated mitogenesis of beta-IR(-/-) cells. In conclusion, independently of glucose, insulin can mediate mitogenesis in fetal pancreatic beta-cell lines. However, in the absence of the insulin receptor, glucose induces beta-cell mitogenesis.

The brown adipose cell: a model for understanding the molecular mechanisms of insulin resistance.

Type 2 diabetes mellitus is a complex metabolic disease that occurs when insulin secretion can no longer compensate insulin resistance in peripheral tissues. At the molecular level, insulin resistance correlates with impaired insulin signalling. This review provides new insights into the molecular mechanisms of insulin action and resistance in brown adipose tissue and pinpoints the role of this tissue in the control of glucose homeostasis. Brown adipocytes are target cells for insulin and IGF-I action, especially during late foetal development when insulin supports survival and promotes both adipogenic and thermogenic differentiation. The main pathway involved in insulin induction of adipogenic differentiation, monitored by fatty acid synthase expression, is the cascade insulin receptor substrate (IRS)-1/phosphatidylinositol 3-kinase (PI3K)/Akt. Glucose transport in these cells is maintained mainly by the activity of GLUT4. Acute insulin treatment stimulates glucose transport largely by mediating translocation of GLUT4 to the plasma membrane, involving the activation of IRS-2/PI3K, and the downstream targets Akt and protein kinase C zeta. Tumour necrosis factor (TNF-alpha) caused insulin resistance on glucose uptake by impairing insulin signalling at the level of IRS-2. Activation of stress kinases and phosphatases by this cytokine contribute to insulin resistance. Furthermore, brown adipocytes are also target cells for rosiglitazone action since they show a high expression of peroxisome proliferator activated receptor gamma, and rosiglitazone increased the expression of the thermogenic uncoupling protein 1. Rosiglitazone ameliorates insulin resistance provoked by TNF-alpha, completely restoring insulin-stimulated glucose uptake in parallel to the insulin signalling cascade. Accordingly, foetal brown adipocytes represent a model for investigating insulin action, as well as for the mechanism by which rosiglitazone increase insulin sensitivity under situations that mimic insulin resistance.

Brown adipose tissue-specific insulin receptor knockout shows diabetic phenotype without insulin resistance.

Although insulin regulates metabolism in both brown and white adipocytes, the role of these tissues in energy storage and utilization is quite different. Recombination technology using the Cre-loxP approach allows inactivation of the insulin receptor in a tissue-specific manner. Mice lacking insulin receptors in brown adipocytes show an age-dependent loss of interscapular brown fat but increased expression of uncoupling protein-1 and -2. In parallel, these mice develop an insulin-secretion defect resulting in a progressive glucose intolerance, without insulin resistance. This model provides direct evidence for not only a role for the insulin receptors in brown fat adipogenesis, the data also suggest a novel role of brown adipose tissue in the regulation of insulin secretion and glucose homeostasis.

[Progressive amyotrophy of a limb as the presenting symptom of anchored spinal cord syndrome with spinal lipoma]. / Amiotrofia progresiva de una extremidad como síntoma de presentación del síndrome de médula anclada con lipoma espina.

OBJECTIVE: We present a case of progressive amyotrophy of a limb as the presenting symptom of the anchored spinal cord syndrome and review the principal clinical features of the syndrome, diagnostic tests which are useful in differentiating it from other conditions and its treatment. CLINICAL CASE: We describe a case of a young woman in whom the spinal cord was anchored by a spinal lipoma. At the onset of the disorder she complained of progressive muscular atrophy of the right leg and difficulty in dorsi-flexion of her right foot. Plain X-ray of the pelvis showed partial agenesis of the right lower hemisacrum and partial sacralization of L5. Lumbosacral CT and MR showed a lipoma to be present within the spinal canal and the thickened filum terminale anchored within the lipoma. During the next five months after diagnosis, the clinical picture worsened with paresia of flexo-extension of the right knee and of flexion of the right foot. Surgical treatment was therefore indicated. CONCLUSIONS: The anchored spinal cord syndrome should be considered in the differential diagnosis of spinal cord disorders presenting in adults, when there are other malformations such as agenesis of the sacrum. Surgical treatment is always indicated when there is evidence of worsening clinical condition.

Association of insulin receptor substrate 1 (IRS-1) y895 with Grb-2 mediates the insulin signaling involved in IRS-1-deficient brown adipocyte mitogenesis.

We have recently generated immortalized fetal brown adipocyte cell lines from insulin receptor substrate 1 (IRS-1) knockout mice and demonstrated an impairment in insulin-induced lipid synthesis as compared to wild-type cell lines. In this study, we investigated the consequences of IRS-1 deficiency on mitogenesis in response to insulin. The lack of IRS-1 resulted in the inability of insulin-stimulated IRS-1-deficient brown adipocytes to increase DNA synthesis and enter into S/G2/M phases of the cell cycle. These cells showed a severe impairment in activating mitogen-activated protein kinase kinase (MEK1/2) and p42-p44 mitogen-activated protein kinase (MAPK) upon insulin stimulation. IRS-1-deficient cells also lacked tyrosine phosphorylation of SHC and showed no SHC-Grb-2 association in response to insulin. The mitogenic response to insulin could be partially restored by enhancing IRS-2 tyrosine phosphorylation and its association with Grb-2 by inhibition of phosphatidylinositol 3-kinase activity through a feedback mechanism. Reconstitution of IRS-1-deficient brown adipocytes with wild-type IRS-1 restored insulin-induced IRS-1 and SHC tyrosine phosphorylation and IRS-1-Grb-2, IRS-1-SHC, and SHC-Grb-2 associations, leading to the activation of MAPK and enhancement of DNA synthesis. Reconstitution of IRS-1-deficient brown adipocytes with the IRS-1 mutant Tyr895Phe, which lacks IRS-1-Grb-2 binding, restored SHC-IRS-1 association and SHC-Grb-2 association. However, the lack of IRS-1-Grb-2 association impaired MAPK activation and DNA synthesis in insulin-stimulated mutant cells. These data provide strong evidence for an essential role of IRS-1 and its direct association with Grb-2 in the insulin signaling pathway leading to MAPK activation and mitogenesis in brown adipocytes.

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.

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.

Epidermal growth factor impairs the cytochrome C/caspase-3 apoptotic pathway induced by transforming growth factor beta in rat fetal hepatocytes via a phosphoinositide 3-kinase-dependent pathway.

Transforming growth factor beta (TGF-beta)-mediated apoptosis is one of the major death processes in the liver. We have previously shown that epidermal growth factor (EGF) is an important survival signal for TGF-beta-induced apoptosis in fetal hepatocytes (Fabregat et al., FEBS Lett 1996;384:14-18). In this work we have studied the intracellular signaling implicated in the protective effect of EGF. We show here that EGF activates p42 and p44 mitogen-activated protein kinases (MAPK). However, mitogen extracellular kinase (MEK) inhibitors do not block the survival effect of EGF. EGF also activates phosphoinositide 3-kinase (PI 3-kinase) and protein kinase B (PKB/AKT) in these cells. The presence of PI 3-kinase inhibitors blocks the protective effect of EGF on cell viability, DNA fragmentation, and caspase-3 activity. We have found that TGF-beta disrupts the mitochondrial transmembrane potential (DeltaPsi(m))( )and activates the release of cytochrome c, this effect being blocked by EGF, via a PI 3-kinase-dependent pathway. A detailed study on bcl-2 superfamily gene expression shows that TGF-beta produces a decrease in the messenger RNA (mRNA) and protein levels of bcl-x(L), an antiapoptotic member of this family, capable of preventing cytochrome c release. EGF is able to maintain bcl-x(L) levels even in the presence of TGF-beta. PI 3-kinase inhibitors completely block the protective effect of EGF on TGF-beta-induced bcl-x(L )down-regulation. We conclude that PI 3-kinase mediates the survival effect of EGF on TGF-beta-induced death by acting upstream from the mitochondrial changes, i.e., preventing bcl-x(L) down-regulation, cytochrome c release, and activation of caspase-3.

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.

Insulin signaling in insulin receptor substrate (IRS)-1-deficient brown adipocytes: requirement of IRS-1 for lipid synthesis.

Immortalized fetal brown adipocyte cell lines have been generated from homozygous (-/-) and heterozygous (+/-) insulin receptor substrate (IRS)-1-deficient mice, as well as from wild-type mice (+/+). Under growing conditions, these cell lines maintained the expression of the adipogenic marker fatty acid synthase and uncoupling protein-1, a tissue-specific thermogenic marker. The IRS-1 (-/-) brown adipocytes lacked IRS-1 protein expression and had a significant increase in IRS-2 protein expression. Insulin-induced tyrosine phosphorylation of IRS-1 was reduced by 50% in heterozygous IRS-1-deficient cells and was totally absent in homozygous cells, while tyrosine phosphorylation of IRS-2 showed a gradual increase. Insulin receptor alpha-subunit protein content and beta-subunit tyrosine kinase activity remained unchanged upon insulin stimulation, regardless of the lack of IRS-1. Brown adipocytes from homozygous IRS-1-deficient mice showed no IRS-1-associated p85alpha subunit of phosphatidylinositol 3-kinase (PI 3-kinase) or IRS-1-associated PI 3-kinase activity in response to insulin, but exhibited enhanced IRS-2-associated p85alpha subunit and IRS-2-associated PI 3-kinase activity. Overall insulin-induced PI 3-kinase activity associated to antiphosphotyrosine immune complexes was decreased by 30% in the homozygous IRS-1-deficient brown adipocytes. Downstream PI 3-kinase, activated Akt (protein kinase B) was decreased by 92% in an insulin-stimulated homozygous IRS-1-deficient brown adipocyte cell line, whereas the expression of Akt was similar in the three cell lines. However, activated p70 S6 kinase (p70s6k) remained unchanged. Although brown adipocyte cell lines showed similar cytosolic lipid content in the presence of 10% fetal calf serum, cytosolic lipid content was reduced in both serum-deprived heterozygous and homozygous IRS-1-deficient cells. Insulin treatment for 24 h doubled the cytosolic lipid content in wild-type and heterozygous IRS-1-deficient brown adipocyte cell lines but failed to increase the cytosolic lipid content in homozygous IRS-1-deficient cells. Our results strongly suggest that IRS-1/PI 3-kinase/Akt activation is an essential requirement for insulin stimulation of lipid synthesis in brown adipocytes.

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.

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.

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.

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.

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.