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.

IGF-I is a mitogen involved in differentiation-related gene expression in fetal rat brown adipocytes.

Fetal rat brown adipocytes at time zero of culture constitute a population of cells of broad spectrum, as estimated by cell size, endogenous fluorescence and lipid content, and show an intrinsic mitogenic competence. They express constitutively early growth-related genes such as c-myc, c-fos, and beta-actin, tissue specific-genes such as the uncoupling protein (UCP) and the lipogenic marker malic enzyme (ME). Fetal brown adipocytes bear a high expression of insulin-like growth factor receptor (IGF-IR), and show a high affinity IGF-I specific-binding to its receptor, and a high number of binding sites per cell. After cell quiescence, insulin-like growth factor I (IGF-I) was as potent as 10% FCS in inducing DNA synthesis, cell number increase, and the entry of cells into the cell-cycle. In addition, IGF-I or 10% FCS for 48 h increased the percentage of [3H]thymidine-labeled nuclei as compared to quiescent cells. Single cell autoradiographic microphotographs show typical multilocular fat droplets brown adipocytes, resulting positive to [3H]thymidine-labeled nuclei in response to IGF-I. IGF-I increased mRNA expression of the early-response genes c-fos (30 min), c-myc (2 and 24 h), and H-ras (4 and 24 h). 10% FCS also increased c-fos and c-myc, but failed to increase H-ras as an early event. IGF-I or 10% FCS, however, similarly increased the mRNA late expression of c-myc, H-ras, c-raf, beta-actin, and glucose 6-phosphate dehydrogenase (G6PD) at 72 h, as compared to quiescent cells. IGF-I or FCS maintained at 24 h or increased at 48 and 72 h UCP mRNA expression. The results demonstrate that IGF-I is a mitogen for fetal rat brown adipocytes, capable of inducing the expression of early and late growth-regulated genes, and of increasing the lipogenic marker ME and the tissue-specific gene UCP, suggesting the involvement of IGF-I in the differentiation as well as in the proliferation processes.

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.

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.

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.

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.

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.

Phosphatidylinositol 3-kinase is a requirement for insulin-like growth factor I-induced differentiation, but not for mitogenesis, in fetal brown adipocytes.

In the present study we have examined the role of phosphatidylinositol 3-kinase (PI 3-kinase) in the insulin-like growth factor I (IGF-I)-signaling pathways involved in differentiation and in mitogenesis in fetal rat brown adipocytes. Activation of PI 3-kinase in response to IGF-I was markedly inhibited by two PI 3-kinase inhibitors (wortmannin and LY294002) in a dose-dependent manner. IGF-I-stimulated glucose uptake was also inhibited by both compounds. The expression of adipogenic-related genes such as fatty acid synthase, malic enzyme, glycerol 3-phosphate dehydrogenase, and acetylcoenzyme A carboxylase induced by IGF-I was totally prevented in the presence of IGF-I and any of those inhibitors, resulting in a marked decrease of the cytoplasmic lipid content. Moreover, the expression of the thermogenic marker uncoupling protein induced by IGF-I was also down-regulated in the presence of wortmannin/LY294002. IGF-I-induced adipogenic- and thermogenic-related gene expression was only partly inhibited by the p70S6k inhibitor rapamycin. In addition, pretreatment of brown adipocytes with either wortmannin or LY294002, but not with rapamycin, blocked protein kinase C zeta activation by IGF-I. In contrast, IGF-I-induced fetal brown adipocyte proliferation was PI 3-kinase-independent. Our results show for the first time an essential requirement of PI 3-kinase in the IGF-I-signaling pathways leading to fetal brown adipocyte differentiation, but not leading to mitogenesis. In addition, protein kinase C zeta seems to be a signaling molecule also involved in the IGF-I differentiation pathways downstream from PI 3-kinase.

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.

IGF-I: a mitogen also involved in differentiation processes in mammalian cells.

The main source of insulin-like growth factor I (IGF-I) postnatally is the liver, under growth hormone stimulation, although IGF-I is already present in embryonic tissues and in fetal serum, when its expression is independent of growth hormone. The extracellular alpha-subunit of the IGF-I receptor (IGF-IR) contains an IGF-I binding domain, and the beta-subunit possesses tyrosine kinase activity, which is greatly enhanced when IGF-I binds to the alpha-subunit and leads to its autophosphorylation. Insulin receptor substrate 1 (IRS-1) is the most well characterized cellular substrate for IGF-I, containing at least 20 potential tyrosine phosphorylation sites. The tyrosine phosphorylated form of IRS-1 acts as a docking protein by associating SH2-containing proteins including the p85 regulatory subunit of phosphatidylinositol-3-kinase (P13-kinase), the protein tyrosine phosphatase SH-PTP2, the SH2- and SH3-containing adaptor protein Nck and the growth factor receptor-bound protein-2 (Grb2/Sem5) protein. Grb2 is found associated with mSOS, a GTP/GDP exchange factor involved in converting the inactive Ras-GDP to the active Ras-GTP. The p85 regulatory subunit of PI3-kinase can be also a direct in vitro substrate of the IGF-IR. Although IRS-1 is the major substrate of the IGF-IR, there is another early phosphotyrosine substrate termed SHC, which also activates Ras via Grb2-mSos complex. Activation of p21-Ras induces a serine/threonine kinase cascade leading to the activation of MAP-kinases. The importance of IGF-I as a mitogen throughout development has been clearly demonstrated in IGF-I and IGF-IR knockout mouse studies and also in transgenic mice over-expressing IGF-I. IGF-I is a mitogen in many cell types in culture such as T lymphocytes, chondrocytes or osteoblasts and it is considered to be a progression factor in mouse fibroblasts. IGF-I is also involved in muscle, neurons and adipogenic differentiation of mesenchymal cells. However, IGF-I induces proliferation and differentiation in fetal brown adipocytes, suggesting that both cellular processes are not necessarily mutually exclusive in fetal cells.

Involvement of Raf-1 kinase and protein kinase C zeta in insulin-like growth factor I-induced brown adipocyte mitogenic signaling cascades: inhibition by cyclic adenosine 3',5'-monophosphate.

In the present study we have examined the signaling cascades involved in insulin-like growth factor I (IGF-I)-induced mitogenesis in fetal rat brown adipocyte primary cultures, a model that constitutively expresses a high number of IGF-I receptors, where IGF-I is a complete mitogen at physiological concentrations. IGF-I rapidly stimulated beta-chain IGF-I receptor autophosphorylation, which peaked at a physiological/mitogenic concentration (1.4 nM) and also stimulated tyrosine phosphorylation of insulin receptor substrate-1 (IRS-1). Tyrosine-phosphorylated IRS-1 bound and subsequently activated phosphatidylinositol 3-kinase by 3.5-fold, whereas the tyrosine-phosphorylated IGF-I receptor was not directly associated with the p85 subunit of the phosphatidylinositol 3-kinase. Moreover, mitogenic concentrations of IGF-I enhanced glucose transport by 2.5-fold. In addition, tyrosine phosphorylation of the 46- and 52-kDa SHC proteins was high in the basal state and doubled after IGF-I treatment, whereas IGF-I enhanced by 4-fold tyrosine phosphorylation of the 66-kDa SHC band. Furthermore, a 2-fold increase in the Ras. GTP active form was induced upon IGF-I stimulation. Downstream from Ras, IGF-I increased both Raf kinase and protein kinase C (PKC) zeta activities by 3.5-fold. (Bu)2cAMP, an inhibitor of IGF-I-induced mitogenesis in fetal brown adipocyte primary cultures, did not block the very early steps of the IGF-I-induced mitogenic cascade, such as IGF-I receptor autophosphorylation, IRS-1 or SHC tyrosine phosphorylation, and Ras activation to its GTP active form. However, (Bu)2cAMP disrupted IGF-I-Raf and IGF-I-PKC zeta signaling pathways by preventing IGF-I-induced Raf-1 kinase and PKC zeta enzymatic activities, respectively. Our results show the first characterization in situ of an IGF-I mitogenic signaling cascade that downstream Ras diverges to the nucleus through two different serine/threonine kinases (Raf-1 kinase and PKC zeta) in mammalian fetal primary cells under physiological conditions. Both kinases represent a point of regulation primarily described for IGF-I-induced, cAMP-inhibited mitogenic pathways.

Alterations in the insulin signaling pathway induced by immortalization and H-ras transformation of brown adipocytes.

In fetal brown adipocyte primary cultures, insulin rapidly (at 5 min) induced tyrosine phosphorylation of the insulin receptor beta-subunit; this effect was maximal at physiological concentrations (1 nM). Insulin also stimulated insulin receptor substrate-1 tyrosine phosphorylation and subsequently activated phosphatidylinositol 3-kinase. Moreover, a 3-fold increase in the Ras.GTP active form and a 6-fold increase in Raf-1 kinase activity were induced after insulin stimulation. An immortalized brown adipocyte cell line (by permanent simian virus 40 large T antigen and pMEXneo cotransfection) showed a reduced maximal responsiveness to insulin in the same range of insulin concentrations studied (1-100 nM). Transformed brown adipocyte cell line (by permanent simian virus 40 large T antigen and pMEXneo H-ras(lys12) cotransfection) developed insulin resistance upstream from Ras, showing an impairment in the insulin receptor autophosphorylation, and in insulin receptor substrate-1 tyrosine phosphorylation and its association with phosphatidylinositol 3-kinase upon treatment with 1 nM insulin, although insulin receptor number and affinity (Kd) remained unaltered. This lack of effect was ameliorated upon treatment with higher insulin concentrations, in a dose-dependent manner. However, downstream from Ras, events such as formation of the Ras.GTP active form, and Raf-1 kinase and 12-O-tetradecanoylphorbol-13-acetate response element-chloramphenicol transferase (transiently transfected) activities were overstimulated, compared with those in primary and immortalized cells, in an insulin-independent manner. Wheat-germ lectin-purified receptors from H-ras(lys12)-transformed brown adipocytes showed a marked phosphorylation in the basal state, which was suppressed by serine-threonine phosphatase pretreatment. Moreover, alkaline phosphatase pretreatment restored the tyrosine kinase activity of the receptor in response to insulin. We conclude that the decreased tyrosine autophosphorylation rate of the insulin receptor from H-ras(lys12)-transformed brown adipocytes is a consequence of its basal serine/threonine phosphorylation, resulting in severe insulin resistance.

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.

Transforming growth factor beta 1 induces differentiation-specific gene expression in fetal rat brown adipocytes.

Fetal rat brown adipocytes show a low number of transforming growth factor beta 1 (TGF-beta 1) binding sites of high affinity, revealing the presence of type I, II and III TGF-beta 1 receptors and a minor-labeled species of approximately 140 kDa. The culture of cells in the presence of TGF-beta 1 induced the expression of the tissue-specific gene uncoupling protein in a dose- and time-dependent manner. In addition, TGF-beta 1 up-regulates the expression of genes involved in adipogenesis such as fatty acid synthase, glycerol 3-phosphate dehydrogenase, malic enzyme and glucose 6-phosphate dehydrogenase, as well as induces the expression of fibronectin (specific target gene for TGF-beta 1). Our results suggest that TGF-beta 1 is a major signal involved in initiating and/or maintaining the thermogenic and adipogenic differentiation of rat fetal brown adipocytes.

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.

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.

Protein kinase D (PKD): a novel target for diacylglycerol and phorbol esters.

A novel serine/threonine protein kinase regulated by phorbol esters and diacylglycerol (named PKD) has been identified. PKD contains a cysteine-rich repeat sequence homologous to that seen in the regulatory domain of protein kinase C (PKC). A bacterially expressed NH2-terminal domain of PKD exhibited high affinity phorbol ester binding activity (Kd = 35 nM). Expression of PKD cDNA in COS cells conferred increased phorbol ester binding to intact cells. The catalytic domain of PKD contains all characteristic sequence motifs of serine protein kinases but shows only a low degree of sequence similarity to PKCs. The bacterially expressed catalytic domain of PKD efficiently phosphorylated the exogenous peptide substrate syntide-2 in serine but did not catalyse significant phosphorylation of a variety of other substrates utilised by PKCs and other major second messenger regulated kinases. PKD expressed in COS cells showed syntide-2 kinase activity that was stimulated by phorbol esters in the presence of phospholipids. We propose that PKD may be a novel component in the transduction of diacylglycerol and phorbol ester signals.

[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.

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.

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.