Activation Of Phosphatidylinositol Research Articles

The vitamin D receptor‐mediated activation of phosphatidylinositol 3‐kinase (PI3Kα) plays a role in the 1α,25‐dihydroxyvitamin D3‐stimulated increase in steroid sulphatase activity in myeloid leukaemic cell lines

In this article we show that 1alpha,25-dihydroxyvitamin D(3) (1alpha,25(OH)(2)D(3)) stimulates the activity of the class IA phosphatidylinositol 3-kinase PI3Kalpha and its downstream target Akt in HL60, U937 and THP-1 myeloid leukaemic cell lines. Furthermore, we show that the classical nuclear vitamin D receptor (VDR(nuc)) is involved in this activation of the PI3K/Akt signalling in these cell lines. We have previously shown that the activity of steroid sulphatase is stimulated in HL60, U937 and THP-1 myeloid leukaemic cell lines by 1alpha,25(OH)(2)D(3) (Hughes et al., [2001] Biochem J 355:361-371; Hughes et al., [2005] J Cell Biochem 94:1175-1189; Hughes and Brown [2006] J Cell Biochem 98:590-617). In this article we show that the 1alpha,25(OH)(2)D(3)-stimulated increase in signalling via the PI3K/Akt pathway plays a role in the increase in steroid sulphatase activity in the HL60 U937 and THP-1 cell lines. We used a variety of pharmacological and biochemical approaches to show that activation of PI3Kalpha mediates the 1alpha,25(OH)(2)D(3)-stimulated increase in steroid sulphatase activity in myeloid leukaemic cells. We also show that the PI3K/Akt dependent activation of NF-kappaB plays a role in the 1alpha,25(OH)(2)D(3)-stimulated increase in steroid sulphatase activity in myeloid leukaemic cells.

K252a Prevents Nigral Dopaminergic Cell Death Induced by 6-Hydroxydopamine through Inhibition of Both Mixed-Lineage Kinase 3/c-Jun NH2-Terminal Kinase 3 (JNK3) and Apoptosis-Inducing Kinase 1/JNK3 Signaling Pathways

It is well documented that the mitogen-activated protein kinase pathway plays a pivotal role in rats with 6-hydroxydopamine (6-OHDA)-induced unilateral lesion in the nigrostriatal system. Our recent studies have shown that mixed-lineage kinase 3 (MLK3) and apoptosis-inducing kinase 1 (ASK1) are all involved in neuronal cell death induced by ischemia, which is mediated by the MLK3/c-Jun NH2-terminal kinase 3 (JNK3) and ASK1/JNK signaling pathway. To investigate whether these pathways are correlated with 6-OHDA-induced lesion as well, we examined the phosphorylation of MLK3, ASK1, and JNK3 in 6-OHDA rats. The results showed that both MLK3 and ASK1 could activate JNK3 and then subsequently enhance the neuronal death through its downstream pathways (i.e., nuclear and non-nuclear pathway). K252a have wide-range effects including Trk inhibition, MLK3 inhibition, and activation of phosphatidylinositol 3 kinase and mitogen-activated protein kinase kinase signaling pathways through interactions with distinct targets and is a well known neuroprotective compound. We found that K252a could protect dopaminergic neurons against cell program death induced by 6-OHDA lesion, and the phenotypes of 6-OHDA rat model treated with K252a were partial rescued. The inhibition of K252a on the activation of MLK3/JNK3 and ASK1/JNK3 provided a link between 6-OHDA lesion and stress-activated kinases. It suggested that both proapoptotic MLK3/JNK3 and ASK1/JNK3 cascade may play an important role in dopaminergic neuronal death in 6-OHDA insult. Thus, the JNK3 signaling may eventually emerge as a prime target for novel therapeutic approaches to treatment of Parkinson disease, and K252a may serve as a potential and important neuroprotectant in therapeutic aspect in Parkinson disease.

Negative feedback regulation of Rac in leukocytes from mice expressing a constitutively active phosphatidylinositol 3-kinase γ

Polarization of chemotaxing cells depends on positive feedback loops that amplify shallow gradients of chemoattractants into sharp intracellular responses. In particular, reciprocal activation of phosphatidylinositol 3-kinases (PI3Ks) and small GTPases like Rac leads to accumulation, at the leading edge, of the PI3K product phosphatidylinositol 3,4,5-trisphosphate (PIP3). Mice carrying a "knockin" allele of the G protein-coupled receptor (GPCR)-activated PI3Kgamma, encoding a plasma membrane-targeted protein appeared normal, but their leukocytes showed GPCR-uncoupled PIP3 accumulation. In vivo, the mutation increased proliferation and decreased apoptosis, leading to leukocytosis and delayed resolution of inflammation in wound healing. Mutant leukocytes showed significantly impaired directional cell migration in response to chemoattractants. Stimulated mutant macrophages did not polarize PIP3 and showed a shortened Rac activation because of enhanced PI3K-dependent activation of RacGAPs. Together with the finding that chemoattractants stimulate a PIP3-dependent GAP activation in wild-type macrophages, these results identify a molecular mechanism involving PI3K- and RacGAP-dependent negative control of Rac that limits and fine-tunes feedback loops promoting cell polarization and directional motility.

Phosphatidylinositol Ether Lipid Analogues That Inhibit AKT Also Independently Activate the Stress Kinase, p38α, through MKK3/6-independent and -dependent Mechanisms

Previously, we identified five active phosphatidylinositol ether lipid analogues (PIAs) that target the pleckstrin homology domain of Akt and selectively induce apoptosis in cancer cells with high levels of Akt activity. To examine specificity, PIAs were screened against a panel of 29 purified kinases. No kinase was inhibited, but one isoform of p38, p38alpha, was uniformly activated 2-fold. Molecular modeling of p38alpha revealed the presence of two regions that could interact with PIAs, one in the activation loop and a heretofore unappreciated region in the upper lobe that resembles a pleckstrin homology domain. In cells, two phases of activation were observed, an early phase that was independent of the upstream kinase MKK3/6 and inhibited by the p38 inhibitor SB203580 and a latter phase that was coincident with MKK3/6 activation. In short term xenograft experiments that employed immunohistochemistry and immunoblotting, PIA administration increased phosphorylation of p38 but not MKK3/6 in tumors in a statistically significant manner. Although PIAs rapidly activated p38 with similar time and dose dependence as Akt inhibition, p38 activation and Akt inhibition were independent events induced by PIAs. Using SB203580 or p38alpha(-/-) cells, we showed that p38alpha is not required for PIA-induced apoptosis but is required for H(2)O(2)- and anisomycin-induced apoptosis. Nonetheless, activation of p38a contributes to PIA-induced apoptosis, because reconstitution of p38a into p38alpha(-/-) cells increased apoptosis. These studies indicate that p38alpha is activated by PIAs through a novel mechanism and show that p38alpha activation contributes to PIA-induced cell death. Independent modulation of Akt and p38alpha could account for the profound cytotoxicity of PIAs.

Activation of phosphatidylinositol 3-kinase/protein kinase B pathway by a vanadyl compound mediates its neuroprotective effect in mouse brain ischemia

We previously reported that orthovanadate composed of vanadate (V5+) activates phosphatidylinositol 3-kinase (PI3K)/protein kinase B (Akt) signaling through inhibition of protein tyrosine phosphatases, thereby eliciting neuroprotection in brain ischemia/reperfusion injury. However, therapeutic doses of orthovanadate are associated with diarrhea due to inhibition of ATPase. By contrast, vanadyl (V4+) organic compounds show low cytotoxicity. Since both vanadate and vanadyl inhibit protein tyrosine phosphatases, we tested whether bis(1-oxy-2-pyridinethiolato)oxovanadium(IV) [VO(OPT)] in a vanadyl form elicits a neuroprotection in brain ischemia. In a mouse transient middle cerebral artery occlusion (MCAO) model, pre- and post-treatments with VO(OPT) significantly reduced infarct volume in a dose-dependent manner. Like orthovanadate, activation of the PI3K/Akt pathway mediated neuroprotective action. VO(OPT) treatment inhibited reduced Akt phosphorylation at Ser-473 following brain ischemia and restored decreased phosphorylation of forkhead box class O (FOXO) family members such as FKHR, FKHRL1, and AFX. Consistent with inhibition of FOXO dephosphorylation, VO(OPT) treatment blocked elevated expression of Fas-ligand, Bim and active caspase-3 24 h after ischemia/reperfusion. Taken together, a vanadyl compound, VO(OPT) elicits neuroprotective effects on brain ischemia/reperfusion injury without apparent side effects.

Flt‐1, but not Flk‐1 mediates hyperpermeability through activation of the PI3‐K/Akt pathway

Vascular endothelial growth factor (VEGF), a potent mediator of endothelial proliferation and migration, has an important role also in brain edema formation during hypoxia and ischemia. VEGF binds to the tyrosine kinase receptors Flt-1 and Flk-1. Yet, their relative importance for hypoxia-induced hyperpermeability is not well understood. We used an in vitro blood-brain barrier (BBB) model consisting of porcine brain microvascular endothelial cells (BMEC) to determine the role of Flt-1 in VEGF-induced endothelial cell (EC) barrier dysfunction. Soluble Flt-1 abolished hypoxia/VEGF-induced hyperpermeability. Furthermore, selective antisense oligonucleotides to Flt-1, but not to Flk-1, inhibited hypoxia-induced permeability changes. Consistent with these data, addition of the receptor-specific homolog placenta-derived growth factor, which binds Flt-1 but not Flk-1, increased endothelial permeability to the same extent as VEGF, whereas adding VEGF-E, a viral VEGF molecule from the orf virus family activating Flk-1 and neuropilin-1, but not Flt-1, did not show any effect. Using the carcinoma submandibular gland cell line (CSG), only expressing Flt-1, it was demonstrated that activation of Flt-1 is sufficient to induce hyperpermeability by hypoxia and VEGF. Hyperpermeability, induced by hypoxia/VEGF, depends on activation of phosphatidylinositol 3-kinase/Akt (PI3-K/Akt), nitric oxide synthase (NOS) and protein kinase G (PKG). The activation of the PI3-K/Akt pathway by hypoxia was confirmed using an in vivo mice hypoxia model. These results demonstrate that hypoxia/VEGF-induced hyperpermeability can be mediated by activation of Flt-1 independently on the presence of Flk-1 and indicate a central role for activation of the PI3-K/Akt pathway, followed by induction of NOS and PKG activity.

Progestin Effects on Breast Cancer Cell Proliferation, Proteases Activation, andin VivoDevelopment of Metastatic Phenotype All Depend on Progesterone Receptor Capacity to Activate Cytoplasmic Signaling Pathways

Accumulating evidence indicates that progestins are involved in controlling mammary gland tumorigenesis. Here, we assessed the molecular mechanisms of progestin action in breast cancer models with different phenotypes. We examined C4HD cells, an estrogen (ER) and progesterone (PR) receptor-positive murine breast cancer model in which progestins exert sustained proliferative response, the LM3 murine metastatic mammary tumor cell line, which lacks PR and ER expression, and human PR null T47D-Y breast cancer cells. In addition to acting as a transcription factor, PR can also function as an activator of signaling pathways. To explore which of these two functions were involved in progestin responses, reconstitution experiments in the PR-negative models were performed with wild-type PR-B, with a DNA binding mutant C587A-PR, and with mutant PR-BmPro, which lacks the ability to activate cytoplasm signaling pathways. We found that in a cell context either ER-positive or -negative, progestins induced cell growth and modulation of matrix metalloproteinases-9 (MMP-9) and -2 (MMP-2), and urokinase-type plasminogen activator (uPA) activities, via MAPK and phosphatidylinositol 3-kinase/Akt pathways, in cells expressing wild-type PR-B or DNA binding mutant C587A-PR. In contrast, in cells expressing mutant PR-BmPro, progestins did not induce growth. We also found that unliganded PR expression conferred breast cancer cells an in vitro less proliferative phenotype, as compared with cells lacking PR expression. Modulation of this behavior occurred when PR was functioning either as transcription factor or as signaling activator. Finally, we for the first time demonstrated that progestins favor development of breast tumor metastasis via PR function as activator of signaling pathways. Our present findings provide mechanistic support to the design of a novel therapeutic intervention in PR-positive breast tumors involving blockage of PR capacity to activate cytoplasmic signaling.

S100B-mediated protection against the pro-apoptotic effects of ethanol on fetal rhombencephalic neurons

Previously, this laboratory demonstrated that ethanol treatment significantly reduces the number of developing serotonin (5-HT) and other fetal rhombencephalic neurons in rats by augmenting apoptosis. Using a 5-HT 1A agonist we were able to attenuate the ethanol-associated reduction and apoptosis of 5-HT and rhombencephalic neurons. The downstream pro-survival effects of 5-HT 1A stimulation were associated with the activation of phosphatidylinositol 3′kinase (PI-3K) and its subsequent up-regulation of specific NF-κB-dependent pro-survival genes. Using an in vitro model, we investigated the hypothesis that S100B, a protein which is released from astrocytes following 5-HT 1A agonist stimulation, can reduce apoptosis in ethanol-treated rat fetal rhombencephalic neurons. We also evaluated whether the anti-apoptotic effects of S100B on fetal rhombencephalic neurons were linked to the activation of the PI-3K → pAkt pro-survival pathway and the expression of two NF-κB-dependent pro-survival genes: XIAP and Bcl-2. Moreover, we determined whether S100B's pro-survival effects were associated with mitogen activated protein kinase kinase (MAPKK) → p42/p44 MAPK. The results of these investigations demonstrated that S100B treatment prevented ethanol-associated apoptosis of fetal rhombencephalic neurons. In addition, it appears that these neuroprotective effects are linked to activation of the PI-3K pathways, because the PI-3K inhibitor LY294002 blocks the neuroprotective effects of S100B. Moreover, S100B increases the formation of pAkt and the up-regulation of two downstream NF-κB-dependent pro-survival genes: XIAP and Bcl-2. Although the MAPKK inhibitor PD98059 reduced the number of surviving neurons in S100B-treated cultures, S100B did not activate MAPKK.

Tissue kallikrein reverses insulin resistance and attenuates nephropathy in diabetic rats by activation of phosphatidylinositol 3-kinase/protein kinase B and adenosine 5'-monophosphate-activated protein kinase signaling pathways.

We previously reported that iv delivery of the human tissue kallikrein (HK) gene reduced blood pressure and plasma insulin levels in fructose-induced hypertensive rats with insulin resistance. In the current study, we evaluated the potential of a recombinant adeno-associated viral vector expressing the HK cDNA (rAAV-HK) as a sole, long-term therapy to correct insulin resistance and prevent renal damage in streptozotocin-induced type-2 diabetic rats. Administration of streptozotocin in conjunction with a high-fat diet induced systemic hypertension, diabetes, and renal damage in rats. Delivery of rAAV-HK resulted in a long-term reduction in blood pressure, and fasting plasma insulin was significantly lower in the rAAV-HK group than in the control group. The expression of phosphatidylinositol 3-kinase p110 catalytic subunit and the levels of phosphorylation at residue Thr-308 of Akt, insulin receptor B, and AMP-activated protein kinases were significantly decreased in organs from diabetic animals. These changes were significantly attenuated after rAAV-mediated HK gene therapy. Moreover, rAAV-HK significantly decreased urinary microalbumin excretion, improved creatinine clearance, and increased urinary osmolarity. HK gene therapy also attenuated diabetic renal damage as assessed by histology. Together, these findings demonstrate that rAAV-HK delivery can efficiently attenuate hypertension, insulin resistance, and diabetic nephropathy in streptozotocin-induced diabetic rats.

Binding of Elongation Factor eEF1A2 to Phosphatidylinositol 4-Kinase β Stimulates Lipid Kinase Activity and Phosphatidylinositol 4-Phosphate Generation

Eukaryotic protein translation elongation factor 1 alpha 2 (eEF1A2) is an oncogene that transforms mammalian cell lines and increases their tumorigenicity in nude mice. Increased expression of eEF1A2 occurs during the development of breast, ovarian, and lung cancer. Here, we report that eEF1A2 directly binds to and activates phosphatidylinositol 4-kinase III beta (PI4KIIIbeta), an enzyme that converts phosphatidylinositol to phosphatidylinositol 4-phosphate. Purified recombinant eEF1A2 increases PI4KIIIbeta lipid kinase activity in vitro, and expression of eEF1A2 in rat and human cells is sufficient to increase overall cellular phosphatidylinositol 4-kinase activity and intracellular phosphatidylinositol 4-phosphate abundance. siRNA-mediated reduction in eEF1A2 expression concomitantly reduces phosphatidylinositol 4-kinase activity. This identifies a physical and functional relationship between eEF1A2 and PI4KIIIbeta.

AMPA protects cultured neurons against glutamate cytotoxicity via activation of phosphatidylinositol-3 kinase/Akt and inactivation of GSK3β and caspase-3

AMPA protects cultured neurons against glutamate cytotoxicity via activation of phosphatidylinositol-3 kinase/Akt and inactivation of GSK3β and caspase-3

Vascular endothelium as a target for endogenous ouabain: studies on the effect of ouabain on human endothelial cells.

It has been suggested that an endogenous inhibitor of the sodium pump, identified as ouabain, contributes to the regulation of blood pressure and the pathogenesis of certain forms of hypertension. Vascular endothelial cells, whose functional integrity is crucial for the maintenance of blood flow and the antithrombotic activity, could be a target for endogenous ouabain. We studied the effect of ouabain on human umbilical vein endothelial cells (HUVEC) and found that nanomolar concentrations of the glycoside have an antiapoptotic activity that is dependent on the activation of phosphatidylinositol 3 kinase (PI-3K) and extracellular signal-regulated kinases (ERKs). At the same concentrations we found that ouabain affects the endocytosis of 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) through the activation of signaling proteins such as Src kinase. This review sumarizes our findings on the effect of ouabain on HUVEC, the signal transduction pathways involved and the significance of these observations on the pathophysiology of endothelial function.

Activation of phosphatidylinositol 3-kinase/Akt limits FGF-2-induced VEGF release in osteoblasts

We previously reported that basic fibroblast growth factor (FGF-2) activates stress-activated protein kinase/c- Jun N-terminal kinase (SAPK/JNK) and p44/p42 mitogen-activated protein (MAP) kinase, resulting in the release of vascular endothelial growth factor (VEGF) in osteoblast-like MC3T3-E1 cells. In the present study, we investigated the role of Akt/protein kinase B in the FGF-2-stimulated VEGF release in these cells. FGF-2 time-dependently induced the phosphorylation of Akt and GSK-3β, a downstream element of Akt. The Akt inhibitor, 1L-6-hydroxymethyl- chiro-inositol 2-( R)-2- O- methyl-3- O-octadecylcarbonate, significantly amplified the FGF-2-induced VEGF release, in a dose-dependent manner between 1 and 70 μM, while it suppressed the FGF-2-induced phosphorylation of GSK-3β. The phosphorylation of Akt induced by FGF-2 was markedly attenuated by wortmannin and LY294002, inhibitors of phosphatidylinositol 3-kinase (PI3-kinase) in osteoblast-like MC3T3-E1 cells. Both wortmannin and LY294002 enhanced the FGF-2-induced VEGF release. In addition, Akt inhibitor had no significant effect on the FGF-2-induced phosphorylation of p44/p42 MAP kinase and SAPK/JNK. Furthermore, the FGF-2-induced Akt phosphorylation was not affected by PD98059, a MEK inhibitor, or SP600125, a SAPK/JNK inhibitor. Taken together, our findings strongly suggest that PI3-kinase/Akt plays an inhibitory role in FGF-2-induced VEGF release in osteoblasts.

Glucose and Insulin Synergistically Activate Phosphatidylinositol 3-Kinase to Trigger Oscillations of Phosphatidylinositol 3,4,5-Trisphosphate in β-Cells

In insulin-secreting beta-cells, activation of phosphatidylinositol 3'-OH-kinase with resulting formation of phosphatidylinositol 3,4,5-trisphosphate (PIP(3)) has been implicated in the regulation of ion channels, insulin secretion, and gene transcription as well as in cell growth and survival, but the kinetics of PIP(3) signals following physiological stimulation of insulin secretion is unknown. Using evanescent wave microscopy and a green fluorescent protein-tagged PIP(3)-binding protein domain for real-time monitoring of plasma membrane PIP(3) concentration in single MIN6 beta-cells, we now demonstrate that glucose stimulation of insulin secretion results in pronounced PIP(3) oscillations via autocrine stimulation of insulin receptors. Glucose lacked effect when insulin secretion was prevented with the hyperpolarizing agent diazoxide, but the sugar dose dependently enhanced the PIP(3) response to maximal insulin stimulation without affecting the rate of PIP(3) degradation. We conclude that glucose is an important co-activator of phosphatidylinositol-3'-OH-kinase and that the plasma membrane PIP(3) concentration in beta-cells undergoes oscillations due to pulsatile release of insulin.

Mutation of the PI3′ Kinase Gene in a Human Colon Carcinoma Cell Line, HCC2998

HCC2998 is a highly differentiated human colon carcinoma cell line, which has been shown to be converted to a poorly differentiated one after expression of a constitutively active phosphatidylinositol 3-kinase (PI3' kinase). These cells express aberrant sizes of a regulatory subunit of PI3' kinase, p85alpha, with molecular weights of 50 and 76 kDa at a very low level. To elucidate how these cells express these proteins, we analyzed mutations within the p85alpha gene. DNA sequencing analysis revealed that these mutant proteins were generated by independent point mutations in the two alleles of the p85alpha gene: one in the coding sequence, and the other in the acceptor sequence for splicing. Introduction of wild-type p85alpha into HCC2998 cells induced slight rounding of the cells and enhancement of mucin secretion. At the same time, a membrane receptor, ErbB3, was phosphorylated on tyrosine, which in turn, binds to PI3' kinase. Since ErbB3 is upstream of PI3' kinase, it is likely that there is an autocrine loop in which PI3' kinase is activated by ErbB3, which may contribute to dedifferentiation of the cells.

γ-H2AX as a Therapeutic Target for Improving the Efficacy of Radiation Therapy

Exposure to ionizing radiation (IR) results in the formation of DNA double strand breaks, resulting in the activation of phosphatidylinositol 3'-kinase-like kinases ATM, ATR and DNK-PKcs. A physiologically important downstream target is the minor histone H2A variant, H2AX, which is rapidly phosphorylated on Ser 139 of the carboxyl tail after IR. Recent work suggests that phosphorylated H2AX (gamma-H2AX) plays an important role in the recruitment and/or retention of DNA repair and checkpoint proteins such as BRCA1, MRE11/RAD50/NBS1 complex, MDC1 and 53BP1. H2AX-/- mouse embryonic fibroblasts are radiation sensitive and demonstrate deficits in repairing DNA damage compared to their wildtype counterparts. Cells treated with peptide inhibitors of gamma-H2AX demonstrate increased radiosensitivity following radiation compared with untreated irradiated cells. Analysis of the kinetics of gamma-H2AX clearance after IR or other DNA damaging agents reveals a correlation between increased gamma-H2AX persistence and unrepaired DNA damage and cell death. These data highlight the potential of post-translational modifications of chromatin as a therapeutic target for enhancing the efficacy of radiotherapy. Therapies that either block gamma-H2AX foci formation by inhibiting upstream kinase activity or that directly inhibit H2AX function may interfere with DNA damage repair processes and warrant further investigation as potential radiosensitizing agents. Agents that increase persistence of gamma-H2AX after IR are likely to increase unrepaired DNA damage.

A Novel Endocytic Mechanism of Epidermal Growth Factor Receptor Sequestration and Internalization

Cells form transient, circular dorsal ruffles or "waves" in response to stimulation of receptor tyrosine kinases, including epidermal growth factor receptor (EGFR) or platelet-derived growth factor receptor. These dynamic structures progress inward on the dorsal surface and disappear, occurring concomitantly with a marked reorganization of F-actin. The cellular function of these structures is largely unknown. Here we show that EGF-induced waves selectively sequester and internalize approximately 50% of ligand-bound EGFR from the cell surface. This process requires receptor phosphorylation, active phosphatidylinositol 3-kinase, and dynamin 2, although clathrin-coated pits or caveolae are not required. Epithelial and fibroblast cells stimulated with EGF sequestered EGFR rapidly into waves that subsequently generated numerous receptor-positive tubular-vesicular structures. Electron microscopy confirmed that waves formed along the dorsal membrane surface and extended numerous tubules into the cytoplasm. These findings characterize a structure that selectively sequesters large numbers of activated EGFR for their subsequent internalization, independent of traditional endocytic mechanisms such as clathrin pits or caveolae.

Follicle-Stimulating Hormone-Induced Aromatase in Immature Rat Sertoli Cells Requires an Active Phosphatidylinositol 3-Kinase Pathway and Is Inhibited via the Mitogen-Activated Protein Kinase Signaling Pathway

Postnatal development and function of testicular Sertoli cells are regulated primarily by FSH. During this early period of development, estrogens play a role in proliferation of somatic cells, which contributes significantly to testicular development. Growth factors like epidermal growth factor (EGF) are produced in the testis and play a role in regulation of estradiol production and male fertility. Although these divergent factors modulate gonadal function, little is known about their mechanism of action in Sertoli cells. The present study investigates the intracellular events that take place down-stream of FSH and EGF receptors in Sertoli cells isolated from immature (10-d-old) rats, and examines which intracellular signals may be involved in their effects on aromatase activity and estradiol production in immature rat Sertoli cells. Primary cultures of rat Sertoli cells were treated with FSH in combination with EGF and signaling pathway-specific inhibitors. Levels of estradiol production, aromatase mRNA (Cyp19a1), and aromatase protein (CYP19A1) were determined. Western blot analysis was performed to determine the effects of FSH and EGF on levels of activated (phosphorylated) AKT1 and p42 ERK2 and p44 ERK1, also named MAPK1 and MAPK3, respectively. The stimulatory actions of FSH on aromatase mRNA, aromatase protein, and estradiol production were blocked by inhibition of the phosphatidylinositol 3-kinase/AKT1 signaling pathway. In contrast, inhibition of ERK signaling augmented the stimulatory effects of FSH on estradiol production, aromatase mRNA, and protein levels. Furthermore, EGF inhibited the expression of aromatase mRNA and protein in response to FSH, and these inhibitory effects of EGF were critically dependent on the activation of the ERK signaling pathway. We conclude that an active phosphatidylinositol 3-kinase /AKT signaling pathway is required for the stimulatory actions of FSH, whereas an active ERK/MAPK pathway inhibits estradiol production and aromatase expression in immature Sertoli cells.

Effect of a 2-day very low-energy diet on skeletal muscle insulin sensitivity in obese type 2 diabetic patients on insulin therapy

This study investigates the molecular mechanisms underlying the blood glucose-lowering effect of a 2-day very low-energy diet (VLED, 1883 kJ/d) in 12 obese (body mass index, 36.3 ± 1.0 kg/m 2 [mean ± SEM]) type 2 diabetic (HbA 1C 7.3% ± 0.4%) patients simultaneously taken off all glucose-lowering therapy, including insulin. Endogenous glucose production (EGP) and glucose disposal ([6,6- 2H 2]-glucose) were measured before and after the VLED in basal and hyperinsulinemic (40 mU/m 2 per minute) euglycemic conditions. Insulin signaling and expression of GLUT-4, FAT/CD36, and triglycerides were assessed in muscle biopsies, obtained before the clamp and after 30 minutes of hyperinsulinemia. Fasting plasma glucose decreased from 11.3 ± 1.3 to 10.3 ± 1.0 mmol/L because of a decreased basal EGP (14.2 ± 1.0 to 11.9 ± 0.7 μmol/kg per minute, P = .009). Insulin-stimulated glucose disposal did not change. No diet effect was found on the expression of the insulin receptor and insulin receptor substrate–1 or on phosphatidylinositol 3′-kinase activity, or on FAT/CD36 expression pattern, GLUT-4 translocation, or triglyceride distribution in either the basal or insulin-stimulated situation. Unexpectedly, basal PKB/Akt phosphorylation on T308 and S473 increased after the diet, at equal protein expression. In conclusion, a 2-day VLED lowers fasting plasma glucose via a decreased basal EGP without an effect on glucose disposal. Accordingly, no changes in activation of phosphatidylinositol 3′-kinase, triglyceride distribution, FAT/CD36 expression, and GLUT-4 translocation were found in skeletal muscle biopsies.

In vitro glucose uptake activity of Aegles marmelos and Syzygium cumini by activation of Glut-4, PI3 kinase and PPARγ in L6 myotubes

The purpose of the present study is to investigate the effect of methanolic extracts of Aegles marmelos and Syzygium cumini on a battery of targets glucose transporter (Glut-4), peroxisome proliferator activator receptor gamma (PPARgamma) and phosphatidylinositol 3' kinase (PI3 kinase) involved in glucose transport. A. marmelos and S. cumini are anti-diabetic medicinal plants being used in Indian traditional medicine. Different solvent extracts extracted sequentially were analysed for glucose uptake activity at each step and methanol extracts were found to be significantly active at 100ng/ml dose comparable with insulin and rosiglitazone. Elevation of Glut-4, PPARgamma and PI3 kinase by A. marmelos and S. cumini in association with glucose transport supported the up-regulation of glucose uptake. The inhibitory effect of cycloheximide on A. marmelos- and S. cumini-mediated glucose uptake suggested that new protein synthesis is required for the elevated glucose transport. Current observation concludes that methanolic extracts of A. marmelos and S. cumini activate glucose transport in a PI3 kinase-dependent fashion.