Amino acids inhibit Agrp gene expression via an mTOR-dependent mechanism

Metabolic fuels act on hypothalamic neurons to regulate feeding behavior and energy homeostasis, but the signaling mechanisms mediating these effects are not fully clear. Rats placed on a low-protein diet (10% of calories) exhibited increased food intake (P < 0.05) and hypothalamic Agouti-related protein (Agrp) gene expression (P = 0.002). Direct intracerebroventricular injection of either an amino acid mixture (RPMI 1640) or leucine alone (1 mug) suppressed 24-h food intake (P < 0.05), indicating that increasing amino acid concentrations within the brain is sufficient to suppress food intake. To define a cellular mechanism for these direct effects, GT1-7 hypothalamic cells were exposed to low amino acids for 16 h. Decreasing amino acid availability increased Agrp mRNA levels in GT1-7 cells (P < 0.01), and this effect was attenuated by replacement of the amino acid leucine (P < 0.05). Acute exposure to elevated amino acid concentrations increased ribosomal protein S6 kinase phosphorylation via a rapamycin-sensitive mechanism, suggesting that amino acids directly stimulated mammalian target of rapamycin (mTOR) signaling. To test whether mTOR signaling contributes to amino acid inhibition of Agrp gene expression, GT1-7 cells cultured in either low or high amino acids for 16 h and were also treated with rapamcyin (50 nM). Rapamycin treatment increased Agrp mRNA levels in cells exposed to high amino acids (P = 0.01). Taken together, these observations indicate that amino acids can act within the brain to inhibit food intake and that a direct, mTOR-dependent inhibition of Agrp gene expression may contribute to this effect.     

Hsp27 inhibits Bax activation and apoptosis via a phosphatidylinositol 3-kinase-dependent mechanism

Hsp27 inhibits mitochondrial injury and apoptosis in both normal and cancer cells by an unknown mechanism. To test the hypothesis that Hsp27 decreases apoptosis by inhibiting Bax, Hsp27 expression was manipulated in renal epithelial cells before transient metabolic stress, an insult that activates Bax, induces mitochondrial injury, and causes apoptosis. Compared with control, enhanced Hsp27 expression inhibited conformational Bax activation, oligomerization, and translocation to mitochondria, reduced the leakage of both cytochrome c and apoptosis-inducing factor, and significantly improved cell survival by >50% after stress. In contrast, Hsp27 down-regulation using RNA-mediated interference promoted Bax activation, increased Bax translocation, and reduced cell survival after stress. Immunoprecipitation did not detect Hsp27-Bax interaction before, during, or after stress, suggesting that Hsp27 indirectly inhibits Bax. During stress, Hsp27 expression prevented the inactivation of Akt, a pro-survival kinase, and increased the interaction between Akt and Bax, an Akt substrate. In contrast, Hsp27 RNA-mediated interference promoted Akt inactivation during stress. Hsp27 up- or down-regulation markedly altered the activity of phosphatidylinositol 3-kinase (PI3-kinase), a major regulator of Akt. Furthermore, distinct PI3-kinase inhibitors completely abrogated the protective effect of Hsp27 expression on Akt activation, Bax inactivation, and cell survival. These data show that Hsp27 antagonizes Bax-mediated mitochondrial injury and apoptosis by promoting Akt activation via a PI3-kinase-dependent mechanism.     

Leptin regulates insulin sensitivity via phosphatidylinositol-3-OH kinase signaling in mediobasal hypothalamic neurons

To investigate whether phosphatidylinositol-3 kinase (PI3K) signaling mediates the metabolic effects of hypothalamic leptin action, adenoviral gene therapy was used to direct expression of leptin receptors to the area of the hypothalamic arcuate nucleus (ARC). This intervention markedly improved insulin sensitivity in genetically obese, leptin-receptor-deficient Koletsky (fa^k/fa^k) rats via a mechanism that was not dependent on reduced food intake but was attenuated by 44% by third-ventricular infusion of the PI3K inhibitor LY294002. Conversely, ARC-directed expression of a constitutively active mutant of protein kinase B (PKB/Akt, an enzyme activated by PI3K) mimicked the insulin-sensitizing effect of restored hypothalamic leptin signaling in these animals, despite having no effect on food intake or body weight. These findings suggest that hypothalamic leptin signaling is an important determinant of glucose metabolism and that the underlying neuronal mechanism involves PI3K.     

LY294002 inhibits monocyte chemoattractant protein-1 expression through a phosphatidylinositol 3-kinase-independent mechanism

The effects of LY294002 (LY29) and wortmannin (WM), inhibitors of phosphatidylinositol 3-kinase (PI3K), on monocyte chemoattractant protein-1 (MCP-1) expression by human umbilical vein endothelial cells were investigated. Complete inhibition of interleukin (IL)-1beta-induced Akt phosphorylation occurred at 50 microM LY29 or 100 nM WM. At these concentrations, LY29, but not WM, significantly inhibited constitutive and IL-1beta-induced MCP-1 expression at both protein and mRNA levels. LY303511 (LY30), an inactive analogue of LY29, also inhibited MCP-1 expression. LY29 and LY30 inhibited activation of nuclear factor-kappaB (NF-kappaB). These results suggest that LY29 inhibits MCP-1 expression at least in part via suppression of NF-kappaB, independent of PI3K, and the structure of LY29 and LY30 may be a novel template for development of new anti-inflammatory drugs.     

Regulation of Id2 gene expression by the insulin-like growth factor I receptor requires signaling by phosphatidylinositol 3-kinase

The Id proteins play an important role in proliferation, differentiation, and tumor development. We report here that Id gene expression can be regulated by the insulin-like growth factor I receptor (IGF-IR), a receptor that also participates in the regulation of cellular proliferation and differentiation. Specifically, we found that the IGF-IR activated by its ligand was a strong inducer of Id2 gene expression in 32D murine hemopoietic cells. This activation was not simply the result of cellular proliferation, as Id2 gene expression was higher in 32D cells stimulated by IGF-I than in cells exponentially growing in interleukin-3. The up-regulation of Id2 gene expression was largely dependent on the presence of insulin receptor substrate-1, a major substrate of the IGF-IR and a potent activator of the phosphatidylinositol 3-kinase (PI3K) pathway. The role of PI3K activity in the up-regulation of Id2 gene expression by the IGF-IR was confirmed by different methods and in different cell types. In 32D cells, the up-regulation of Id2 gene expression by the PI3K pathway correlated with interleukin-3 independence and inhibition of differentiation.     

TGF-beta1 inhibits BRCA1 expression through a pathway that requires pRb

TGF-beta1 inhibits BRCA1 expression, which contradicts the model that TGF-beta1 prevents carcinogenesis by activating tumor suppressor genes. To resolve this apparent contradiction, we examined BRCA1 expression in Mv1Lu cells, a well-established model system for studying the TGF-beta1 tumor suppressor pathway. We found that inactivation of pRb by the papillomavirus type 16 E7 protein increased BRCA1 expression and abolished the ability of TGF-beta1 to inhibit BRCA1 expression. We conclude that TGF-beta1 inhibits BRCA1 expression through a pathway that requires pRb. We propose a model to explain the inhibition of BRCA1 as a target in the TGF-beta1 tumor suppressor signaling pathway. Our results suggest that the tumor suppressor functions of BRCA1 are initiated by the inactivation of pRb, and therefore that the activation of pRb by TGF-beta1 might alleviate the requirement for BRCA1 function.     

Leptin constrains phospholipase C-protein kinase C-induced insulin secretion via a phosphatidylinositol 3-kinase-dependent pathway

Leptin-deficient Lep(ob)/Lep(ob)mice hypersecrete insulin in response to acetylcholine stimulation of the phospholipase C-protein kinase C (PLC-PKC) pathway, and leptin constrains this hypersecretion. Leptin has been reported to activate phosphatidylinositol 3-kinase (PI 3-K) and subsequently phosphodiesterase (PDE) to impair protein kinase A (PKA)-induced insulin secretion from cultured islets of neonatal rats. We determined if PKA-induced insulin secretion was also hyperresponsive in islets from Lep(ob)/Lep(ob)mice, and if leptin impaired this pathway in islets from these mice. Additionally, the possible role for PI 3-K and PDE in leptin-induced control of acetylcholine-induced insulin secretion was examined. Stimulation of insulin secretion with GLP-1, forskolin (an activator of adenylyl cyclase), or IBMX (an inhibitor of PDE) did not cause hypersecretion of insulin from islets of young Lep(ob)/Lep(ob)mice, and leptin did not inhibit GLP-1-induced insulin secretion from islets of these mice. Inhibition of PDE with IBMX also did not block leptin-induced inhibition of acetylcholine-mediated insulin secretion from islets of Lep(ob)/Lep(ob)mice. But, preincubation of islets with wortmannin, an inhibitor of PI 3-K activity, blocked the ability of leptin to constrain acetylcholine-induced insulin secretion from islets of Lep(ob)/Lep(ob)mice. We conclude that the capacity of the PKA pathway to stimulate insulin secretion is not increased in islets from young Lep(ob)/Lep(ob)mice, and that leptin does not regulate this pathway in islets from mice. Leptin may stimulate PI 3-K to constrain PLC-PKC-induced insulin secretion from islets of Lep(ob)/Lep(ob)mice.     

Protein kinase C inhibits ROMK1 channel activity via a phosphatidylinositol 4,5-bisphosphate-dependent mechanism

The activity of apical K(+) channels in cortical collecting duct (CCD) is stimulated and inhibited by protein kinase A (PKA) and C (PKC), respectively. Direct interaction between phosphatidylinositol 4,5-bisphosphate (PIP(2)) and the cloned CCD K(+) channel, ROMK1, is critical for channel opening. We have found previously that phosphorylation of ROMK1 by PKA increases affinity of the channel for PIP(2) and mutation of PKA sites reduces the affinity of ROMK1 for PIP(2). In this study we investigate the molecular mechanism for PKC regulation of ROMK and report that mutants of ROMK1 with reduced PIP(2) affinity exhibit an increased sensitivity to inhibition by phorbol myristate acetate (PMA). The effect of PMA can be prevented by pretreatment with calphostin-C. Activation of PKC by carbachol in Xenopus oocytes co-expressing M1 muscarinic receptors also causes inhibition of the channels. Calphostin-C prevents carbachol-induced inhibition, suggesting that activation of PKC is necessary for inhibition of the channels. PMA reduces open probability of the channel in cell-attached patch clamp recordings. After inhibition by PMA in cell-attached recordings, application of PIP(2) to the cytoplasmic face of excised inside-out membranes restores channel activity. PMA reduces PIP(2) content in oocyte membrane and calphostin-C prevents the reduction. These results suggest that reduction of membrane PIP(2) content contributes to the inhibition of ROMK1 channels by PKC. This mechanism may underscore the inhibition of K(+) secretion in CCD by hormones that activate PKC.     

Neuronal suppressor of cytokine signaling-3 deficiency enhances hypothalamic leptin-dependent phosphatidylinositol 3-kinase signaling

Suppressor of cytokine signaling-3 (SOCS3) is thought to be involved in the development of central leptin resistance and obesity by inhibiting STAT3 pathway. Because phosphatidylinositol 3-kinase (PI3K) pathway plays an important role in transducing leptin action in the hypothalamus, we examined whether SOCS3 exerted an inhibition on this pathway. We first determined whether leptin sensitivity in the hypothalamic PI3K pathway was increased in brain-specific Socs3-deficient (NesKO) mice. In NesKO mice, hypothalamic insulin receptor substrate-1 (IRS1)-associated PI3K activity was significantly increased at 30 min and remained elevated up to 2 h after leptin intraperitoneal injection, but in wild-type (WT) littermates, the significant increase was only at 30 min. Hypothalamic p-STAT3 levels were increased up to 5 h in NesKO as opposed to 2 h in WT mice. In food-restricted WT mice with reduced body weight, leptin increased hypothalamic PI3K activity only at 30 min, and p-STAT3 levels at 30-120 min postinjection. These results suggest increased leptin sensitivity in both PI3K and STAT3 pathways in the hypothalamus of NesKO mice, which was not due to a lean phenotype. In the next experiment with a clonal hypothalamic neuronal cell line expressing proopiomelanocortin, we observed that whereas leptin significantly increased IRS1-associated PI3K activity and p-JAK2 levels in cells transfected with control vector, it failed to do so in SOCS3-overexpressed cells. Altogether, these results imply a SOCS3 inhibition of the PI3K pathway of leptin signaling in the hypothalamus, which may be one of the mechanisms behind the development of central leptin resistance and obesity.     

Respiratory syncytial virus inhibits granulocyte apoptosis through a phosphatidylinositol 3-kinase and NF-kappaB-dependent mechanism

Respiratory syncytial virus (RSV) is a common cause of lower respiratory tract disease in children. It is associated with increased neutrophil numbers in the airway. In this study, we assessed whether this ssRNA virus can directly influence granulocyte longevity. By culturing RSV with granulocytes, it was observed that virus delays both constitutive neutrophil and eosinophil apoptosis. Using pharmacological inhibitors, the RSV-induced delay in neutrophil apoptosis was found to be dependent on both PI3K and NF-kappaB, but not p38 MAPK or MEK1/MEK2 activation. Using blocking Abs and a reporter cell line, we were able to exclude TLR4 as the receptor responsible for mediating RSV-induced delay in neutrophil apoptosis. The antiapoptotic effect was abrogated by preincubation with the lysosomotropic agent chloroquine, indicating the requirement for endolysosomal internalization. Furthermore, addition of ssRNA, a ligand for the intracellular TLR7/TLR8, also inhibited neutrophil apoptosis, suggesting that intracellular TLRs could be involved in induction of the antiapoptotic effect. Using the BioPlex cytokine detection assay (Bio-Rad), we found that IL-6 was present in supernatants from RSV-exposed neutrophils. IL-6 was found to inhibit neutrophil apoptosis, suggesting that there is an autocrine or paracrine antiapoptotic role for IL-6. Finally, RSV treatment of neutrophils resulted in increased expression of the antiapoptotic Bcl-2 protein Mcl-1. Taken together, our findings suggest involvement of multiple intracellular mechanisms responsible for RSV-induced survival of granulocytes and point toward a role for intracellular TLRs in mediating these effects.     

Leptin induces macrophage lipid body formation by a phosphatidylinositol 3-kinase- and mammalian target of rapamycin-dependent mechanism

Leptin is an adipocyte-derived hormone/cytokine that links nutritional status with neuroendocrine and immune functions. Lipid bodies (lipid droplets) are emerging as dynamic organelles with roles in lipid metabolism and inflammation. Here we investigated the roles of leptin in signaling pathways involved in cytoplasmic lipid body biogenesis and leukotriene B(4) synthesis in macrophages. Our results demonstrated that leptin directly activated macrophages and induced the formation of adipose differentiation-related protein-enriched lipid bodies. Newly formed lipid bodies were sites of 5-lipoxygenase localization and correlated with an enhanced capacity of leukotriene B(4) production. We demonstrated that leptin-induced macrophage activation was dependent on phosphatidylinositol 3-kinase (PI3K) activity, since the lipid body formation was inhibited by LY294002 and was absent in the PI3K knock-out mice. Leptin induces phosphorylation of p70(S6K) and 4EBP1 key downstream signaling intermediates of the mammalian target of rapamycin (mTOR) pathway in a rapamycin-sensitive mechanism. The mTOR inhibitor, rapamycin, inhibited leptin-induced lipid body formation, both in vivo and in vitro. In addition, rapamycin inhibited leptin-induced adipose differentiation-related protein accumulation in macrophages and lipid body-dependent leukotriene synthesis, demonstrating a key role for mTOR in lipid body biogenesis and function. Our results establish PI3K/mTOR as an important signaling pathway for leptin-induced cytoplasmic lipid body biogenesis and adipose differentiation-related protein accumulation. Furthermore, we demonstrate a previously unrecognized link between intracellular (mTOR) and systemic (leptin) nutrient sensors in macrophage lipid metabolism. Leptin-induced increased formation of cytoplasmic lipid bodies and enhanced inflammatory mediator production in macrophages may have implications for obesity-related cardiovascular diseases.     

Tauroursodeoxycholic acid prevents amyloid-beta peptide-induced neuronal death via a phosphatidylinositol 3-kinase-dependent signaling pathway

Tauroursodeoxycholic acid (TUDCA), an endogenous bile acid, modulates cell death by interrupting classic pathways of apoptosis. Amyloid-beta (Abeta) peptide has been implicated in the pathogenesis of Alzheimer's disease, where a significant loss of neuronal cells is thought to occur by apoptosis. In this study, we explored the cell death pathway and signaling mechanisms involved in Abeta-induced toxicity and further investigated the anti-apoptotic effect(s) of TUDCA. Our data show significant induction of apoptosis in isolated cortical neurons incubated with Abeta peptide. Apoptosis was associated with translocation of pro-apoptotic Bax to the mitochondria, followed by cytochrome c release, caspase activation, and DNA and nuclear fragmentation. In addition, there was almost immediate but weak activation of the serine/threonine protein kinase Akt. Inhibition of the phosphatidylinositide 3 prime-OH kinase (PI3K) pathway with wortmannin did not markedly affect Abeta-induced cell death, suggesting that this signaling pathway is not crucial for Abeta-mediated toxicity. Notably, co-incubation with TUDCA significantly modulated each of the Abeta-induced apoptotic events. Moreover, wortmannin decreased TUDCA protection against Abeta-induced apoptosis, reduced Akt phosphorylation, and increased Bax translocation to mitochondria. Together, these findings indicate that Abeta-induced apoptosis of cortical neurons proceeds through a Bax mitochondrial pathway. Further, the PI3K signaling cascade plays a role in regulating the anti-apoptotic effects of TUDCA.     

Cyclin-mediated inhibition of muscle gene expression via a mechanism that is independent of pRB hyperphosphorylation.

It was recently demonstrated that ectopic expression of cyclin D1 inhibits skeletal muscle differentiation and, conversely, that expression of cyclin-dependent kinase (cdk) inhibitors facilitates activation of this differentiation program (S. S. Rao, C. Chu, and D. S. Kohtz, Mol. Cell. Biol. 14:5259-5267, 1994; S. S. Rao and D. S. Kohtz, J. Biol. Chem. 270:4093-4100, 1995; S. X. Skapek, J. Rhee, D. B. Spicer, and A. B. Lassar, Science 267:1022-1024, 1995). Here we demonstrate that cyclin D1 inhibits muscle gene expression without affecting MyoD DNA binding activity. Ectopic expression of cyclin D1 inhibits muscle gene activation by both MyoD and myogenin, including a mutated form of myogenin in which two potential inhibitory cdk phosphorylation sites are absent. Because the retinoblastoma gene product, pRB, is a known target for cyclin D1-cdk phosphorylation, we determined whether cyclin D1-mediated inhibition of myogenesis was due to hyperphosphorylation of pRB. In pRB-deficient fibroblasts, the ability of MyoD to activate the expression of muscle-specific genes requires coexpression of ectopic pRB (B. G. Novitch, G. J. Mulligan, T. Jacks, and A. B. Lassar, J. Cell Biol., 135:441-456, 1996). In these cells, the expression of cyclins A and E can lead to pRB hyperphosphorylation and can inhibit muscle gene expression. The negative effects of cyclins A or E on muscle gene expression are, however, reversed by the presence of a mutated form of pRB which cannot be hyperphosphorylated. In contrast, cyclin D1 can inhibit muscle gene expression in the presence of the nonhyperphosphorylatable form of pRB. On the basis of these results we propose that G1 cyclin-cdk activity blocks the initiation of skeletal muscle differentiation by two distinct mechanisms: one that is dependent on pRB hyperphosphorylation and one that is independent of pRB hyperphosphorylation.     

VACM-1, a cul-5 gene,inhibits cellular growth by a mechanism that involves MAPK and p53 signaling pathways

Vasopressin-activated Ca²⁺-mobilizing (VACM)-1 gene product is a 780-amino acid membrane protein that shares sequence homology with cullins, a family of genes involved in the regulation of cell cycle. However, when expressed in vitro, VACM-1 attenuates basal and vasopressin- and forskolin-induced cAMP production. Mutating the PKA-dependent phosphorylation site in the VACM-1 sequence (^S730AVACM-1) prevents this inhibitory effect. To further examine the biological role of VACM-1, we studied the effect of VACM-1 and ^S730AVACM-1 proteins on cellular proliferation and gene expression in Chinese hamster ovary and COS-1 cells. Cellular proliferation of VACM-1-expressing cell lines was significantly lower compared with that of the vector-transfected cells, whereas it was significantly increased in ^S730AVACM-1-derived cell lines. Furthermore, expression of VACM-1 but not ^S730AVACM-1 protein retarded cytokinesis and prevented MAPK phosphorylation. Screening with the Human PathwayFinder-1 GEArray system and subsequent Western blot analysis demonstrated that VACM-1 induces p53 mRNA and protein expression. In summary, VACM-1 inhibits cellular growth by a mechanism that involves cAMP, MAPK phosphorylation, and p53 expression. mitogen-activated protein kinase; cytokinesis; vasopressin-activated calcium-mobilizing receptor     

Three-dimensional collagen regulates collagen gene expression by a mechanism that requires serine/threonine kinases and is independent of mechanical contraction

Integrin alpha1beta1, one of the cellular collagen receptors, can participate in the regulation of collagen accumulation by acting as a negative feedback regulator. The molecular mechanism behind this phenomenon has been unknown. We have plated cells inside three-dimensional collagen and analyzed a set of chemical inhibitors for various signal transduction pathways. Only two wide-spectrum serine/threonine kinase inhibitors, H-7 and iso-H-7 could prevent the down-regulation of alpha1(I) collagen mRNA levels in cells exposed to three-dimensional collagen. In monolayer iso-H-7 slightly down-regulated collagen gene expression, indicating that inside collagen it affected integrin signaling rather than having a direct stimulatory effect on collagen mRNA levels. The effect of iso-H-7 was not dependent on its ability to inhibit protein kinases A, C, or G. H-7 and iso-H-7 could also inhibit collagen gel contraction, but this mechanism was independent of collagen gene regulation. Three-dimensional collagen could also up-regulate the mRNA levels of several matrix metalloproteinases (MMPs) but H-7 and iso-H-7 had no effect on the regulation of MMP genes. Our data indicate that three-dimensional collagenous matrix regulates distinct cellular signaling pathways and that collagen gene regulation is independent of the other effects of the matrix.     

Leptin inhibits bone formation through a hypothalamic relay: a central control of bone mass

Gonadal failure induces bone loss while obesity prevents it. This raises the possibility that bone mass, body weight, and gonadal function are regulated by common pathways. To test this hypothesis, we studied leptin-deficient and leptin receptor-deficient mice that are obese and hypogonadic. Both mutant mice have an increased bone formation leading to high bone mass despite hypogonadism and hypercortisolism. This phenotype is dominant, independent of the presence of fat, and specific for the absence of leptin signaling. There is no leptin signaling in osteoblasts but intracerebroventricular infusion of leptin causes bone loss in leptin-deficient and wild-type mice. This study identifies leptin as a potent inhibitor of bone formation acting through the central nervous system and therefore describes the central nature of bone mass control and its disorders.