Journal of Molecular Endocrinology recent issues

Thyroid hormone receptors regulate adipogenesis and carcinogenesis via crosstalk signaling with peroxisome proliferator-activated receptors

Lu, C., Cheng, S.-Y. — Fri, 12 Feb 2010 01:40:13 PST

Peroxisome proliferator-activated receptors (PPARs) and thyroid hormone receptors (TRs) are members of the nuclear receptor superfamily. They are ligand-dependent transcription factors that interact with their cognate hormone response elements in the promoters to regulate respective target gene expression to modulate cellular functions. While the transcription activity of each is regulated by their respective ligands, recent studies indicate that via multiple mechanisms PPARs and TRs crosstalk to affect diverse biological functions. Here, we review recent advances in the understanding of the molecular mechanisms and biological impact of crosstalk between these two important nuclear receptors, focusing on their roles in adipogenesis and carcinogenesis.

The regulatory domain of protein kinase C delta positively regulates insulin receptor signaling

Fri, 12 Feb 2010 01:40:13 PST

Protein kinase C delta (PKC) is induced by insulin to rapidly associate with insulin receptor (IR) and upregulates insulin signaling. We utilized specific JM and CT receptor domains and chimeras of PKC and PKC regulatory and catalytic domains to elucidate which components of PKC are responsible for positive regulatory effects of PKC on IR signaling. Studies were performed on L6 and L8 skeletal muscle myoblasts and myotubes. PKC was preferentially bound to the JM domain of IR, and insulin stimulation increased this binding. Both PKC/ and PKC/ chimeras (regulatory/catalytic) were bound preferentially to the JM but not to the CT domain of IR. Although IR–PKC binding was higher in cells expressing either the PKC/ or PKC/ chimera than in control cells, upregulation of IR signaling was observed only in PKC/ cells. Thus, in response to insulin increases in tyrosine phosphorylation of IR and insulin receptor substrate-1, downstream signaling to protein kinase B and glycogen synthase kinase 3 (GSK3) and glucose uptake were greater in cells overexpressing PKC/ and the PKC/ domains than in cells expressing the PKC/ domains. Basal binding of Src to PKC was higher in both PKC/- and PKC/-expressing cells compared to control. Binding of Src to IR was decreased in PKC/ cells but remained elevated in the PKC/ cells in response to insulin. Finally, insulin increased Src activity in PKC/-expressing cells but decreased it in PKC/-expressing cells. Thus, the regulatory domain of PKC via interaction with Src appears to determine the role of PKC as a positive regulator of IR signaling in skeletal muscle.

Visfatin regulates insulin secretion, insulin receptor signalling and mRNA expression of diabetes-related genes in mouse pancreatic {beta}-cells

Fri, 12 Feb 2010 01:40:13 PST

The role of the adipocyte-derived factor visfatin in metabolism remains controversial, although some pancreatic β-cell-specific effects have been reported. This study investigated the effects of visfatin upon insulin secretion, insulin receptor activation and mRNA expression of key diabetes-related genes in clonal mouse pancreatic β-cells. β-TC6 cells were cultured in RPMI 1640 and were subsequently treated with recombinant visfatin. One-hour static insulin secretion was measured by ELISA. Phospho-specific ELISA and western blotting were used to detect insulin receptor activation. Real-time SYBR Green PCR array technology was used to measure the expression of 84 diabetes-related genes in both treatment and control cells. Incubation with visfatin caused significant changes in the mRNA expression of several key diabetes-related genes, including marked up-regulation of insulin (9-fold increase), hepatocyte nuclear factor (HNF)1β (32-fold increase), HNF4 (16-fold increase) and nuclear factor B (40-fold increase). Significant down-regulation was seen in angiotensin-converting enzyme (–3.73-fold) and UCP2 (–1.3-fold). Visfatin also caused a significant 46% increase in insulin secretion compared to control (P<0.003) at low glucose, and this increase was blocked by co-incubation with the specific nicotinamide phosphoribosyltransferase inhibitor FK866. Both visfatin and nicotinamide mononucleotide induced activation of both insulin receptor and extracellular signal-regulated kinase (ERK)1/2, with visfatin-induced insulin receptor/ERK1/2 activation being inhibited by FK866. We conclude that visfatin can significantly regulate insulin secretion, insulin receptor phosphorylation and intracellular signalling and the expression of a number of β-cell function-associated genes in mouse β-cells.

Intermittent high glucose exacerbates the aberrant production of adiponectin and resistin through mitochondrial superoxide overproduction in adipocytes

Sun, J., Xu, Y., Deng, H., Sun, S., Dai, Z., Sun, Y. — Fri, 12 Feb 2010 01:40:13 PST

Hypoadiponectinemia and hyperresistinemia may be important in mediating signals from adipocytes to insulin-sensitive tissue and vasculature. However, the mechanism that mediates the aberrant production of adipokines remains poorly understood. In this study, we have investigated the effect of intermittent high glucose on the expression of adiponectin and resistin, and the production of 8-hydroxydeoxyguanosine (8-OHdG) and nitrotyrosine in the adipocytes, either in the presence or in the absence of Mn(III) tetrakis(4-benzoic acid) porphyrin chloride (MnTBAP) or thenoyltrifluoroacetone (TTFA). 3T3-L1 adipocytes were incubated for 72 h in media containing different glucose concentrations: 5 mmol/l, 20 mmol/l, 5 mmol/l alternating with 20 mmol/l glucose, with or without MnTBAP and TTFA. We measured the expression of resistin and adiponectin. The production of nitrotyrosine and 8-OHdG as oxidative stress parameter was measured. Both constant and intermittent high glucose significantly suppressed the expression and secretion of adiponectin, and increased expression and secretion of resistin in mature adipocytes compared to normal glucose conditions. However, these effects were significantly greater under intermittent high glucose conditions compared to constant high glucose. The levels of nitrotyrosine and 8-OHdG were significantly elevated under both intermittent and constant high glucose conditions, the effect being greater under intermittent high glucose. In addition, the antioxidants MnTBAP or TTFA reversed the aberrant production of adiponectin and resistin, as well as overproduction of nitrotyrosine and 8-OHdG in adipocytes induced by constant or intermittent high glucose. Intermittent high glucose exacerbates the aberrant production of adiponectin and resistin through reactive oxygen species overproduction at the mitochondrial transport chain level in adipocytes, indicating that glycemic variability has important pathological effects on the secretion of adipokines.

Short form 1b human prolactin receptor down-regulates expression of the long form

Tan, D., Walker, A. M — Fri, 12 Feb 2010 01:40:13 PST

Alternative splicing produces different human prolactin (PRL) receptors. These include a long form (LF) and two short forms (SF1a and SF1b). The SFs of the receptor can act as dominant negatives for PRL effector function through the LF. This is proposed to be due to LF–SF heterodimerization and resultant interference with LF–LF dimer signaling. We, along with others, have provided evidence for LF–SF heterodimerization of the human receptors in support of this mechanism, along with others. However, to further investigate the ways SF may influence LF function, we co-transfected human embryonic kidney 293 cells with vectors coding for tagged (green fluorescent protein (GFP) or luciferase) LF alone or plus untagged SF1b and measured LF-GFP intensity, LF-luciferase activity, and LF mRNA 48 h later. Equal amounts of SF1b cDNA decreased LF-GFP fluorescence intensity, LF-luciferase activity, and LF mRNA by 80–100%. Similar co-transfections with untagged LF had no significant effect on tagged LF expression. Use of hygromycin showed degradation of already formed protein was the same for LF-luciferase alone and LF-luciferase with SF1b. Inhibition of mRNA synthesis, on the other hand, showed that SF1b expression accelerated LF mRNA degradation two- to three-fold. SF1b also down-regulated expression of endogenous LF mRNA in T47D breast cancer cells and opposed an increase in cell number resulting from transfection with extra LF alone. These results demonstrate a previously unrecognized mechanism whereby SF1b affects the end result of signaling through the LF receptor. The effects on cell number also support the concept that the LF:SF1b ratio may be relevant to tumor growth.

Signal transduction of the CB1 cannabinoid receptor

Turu, G., Hunyady, L. — Thu, 14 Jan 2010 17:51:21 PST

The CB₁ cannabinoid receptor (CB₁R) is the major cannabinoid receptor in neuronal cells and the brain, but it also occurs in endocrine cells and other peripheral tissues. CB₁R is a member of the superfamily of G-protein-coupled receptors (GPCRs), which are characterized by seven transmembrane helices. The major mediators of CB₁R are the G proteins of the G_i/o family, which inhibit adenylyl cyclases in most tissues and cells, and regulate ion channels, including calcium and potassium ion channels. Regulation of ion channels is an important component of neurotransmission modulation by endogenous cannabinoid compounds released in response to depolarization and Ca²⁺-mobilizing hormones. However, evidence exists that CB₁Rs can also stimulate adenylyl cyclase via G_s, induce receptor-mediated Ca²⁺ fluxes and stimulate phospholipases in some experimental models. Stimulation of CB₁R also leads to phosphorylation and activation of mitogen-activated protein kinases (MAPK), such as p42/p44 MAPK, p38 MAPK and c-Jun N-terminal kinase, which can regulate nuclear transcription factors. Activated and phosphorylated CB₁Rs also associate with β-arrestin molecules, which can induce the formation of signalling complexes and participate in the regulation of GPCR signalling. Recent data also suggest that CB₁Rs can form homo- and heterodimers/oligomers, and the altered pharmacological properties of these receptor complexes may explain the pharmacological differences observed in various tissues.

AMPK as a mediator of hormonal signalling

Lim, C. T., Kola, B., Korbonits, M. — Thu, 14 Jan 2010 17:51:21 PST

AMP-activated protein kinase (AMPK) is a key molecular player in energy homeostasis at both cellular and whole-body levels. AMPK has been shown to mediate the metabolic effects of hormones such as leptin, ghrelin, adiponectin, glucocorticoids and insulin as well as cannabinoids. Generally, activated AMPK stimulates catabolic pathways (glycolysis, fatty acid oxidation and mitochondrial biogenesis) and inhibits anabolic pathways (gluconeogenesis, glycogen, fatty acid and protein synthesis), and has a direct appetite-regulating effect in the hypothalamus. Drugs that activate AMPK, namely metformin and thiazolidinediones, are often used to treat metabolic disorders. Thus, AMPK is now recognised as a potential target for the treatment of obesity and associated co-morbidities.

Identification of a region in the human IRS2 promoter essential for stress induced transcription depending on SP1, NFI binding and ERK activation in HepG2 cells

Thu, 14 Jan 2010 17:51:21 PST

Recent studies have discovered changes in the insulin-/IGF1 signaling affecting glucose metabolism and the molecular pathogenesis of human hepatocellular cancer. Insulin/IGF1 receptor mediates its intracellular effects by recruitment of one out of the four different insulin receptor substrates (IRS). To investigate mechanisms of IRS2 expression, we analyzed transcriptional regulation of IRS2 in human HepG2 cells. We identified a region 688 bp upstream of the translation start codon responsible for ~90% of basal human IRS2 promoter activity in HepG2 cells, and confirmed binding of specificity protein 1 (also called Sp1 transcription factor, SP1) and nuclear factor 1 (NFI) in this region. Mutation of both SP1 and NFI binding sites or inhibition of extracellular signal regulated kinase (ERK) suppressed IRS2 promoter activity almost completely, revealing a major role of MAP kinases (MAPK) for IRS2 transcription. Activating this cascade with oxidative stress increased IRS2 promoter activity and endogenous IRS2 expression substantially. IRS2 promoter activity rose even more after additional inhibition of p38MAPK indicating an inhibitory effect of p38MAPK on ERK mediated IRS2 transcription. Activation of the MAPK pathway using interleukin 1, beta (IL1B) increased IRS2 promoter activity similar to oxidative stress. In contrast IL1B decreases and inhibition of the MAPK pathway increases IRS1 promoter activity revealing opposed effects of IL1B and ERK on the expression of different IRS proteins. In conclusion we discovered a specific region (–688 to –611 bp) in the IRS2 promoter essential for basal promoter activity and oxidative stress induced transcription depending on ERK activation and SP1 and NFI binding in human hepatocytes.

Epidermal growth factor and hepatocyte growth factor cooperate to enhance cell proliferation, scatter, and invasion in murine mammary epithelial cells

Accornero, P., Miretti, S., Starvaggi Cucuzza, L., Martignani, E., Baratta, M. — Thu, 14 Jan 2010 17:51:21 PST

The development of the mammary gland requires an integrated response to specific growth factors and steroid hormones. Hepatocyte growth factor (HGF) and its tyrosine kinase receptor, MET, are expressed and temporally regulated during mammary development and differentiation. Epidermal growth factor receptor (EGFR) and its ligands have also been implicated in mammary gland growth and morphogenesis. Since both cytokines seem to exert a morphogenic program in this tissue, we have investigated the possible concerted action of EGF and HGF on the HC11 cell line, a widely used model of nontumorigenic mammary cells. Western blot analysis indicated that HC11 expressed MET and EGFR, and showed ERK1/2 and AKT activation following HGF or EGF treatment. Analysis by real-time PCR and western blot showed that after an EGF but not HGF or insulin-like growth factor-I treatment, HC11 mammary cells exhibited an increase in MET expression at both the mRNA and protein levels, which was dependent on the AKT pathway. Simultaneous treatment with HGF and EGF increased proliferation, scatter, and invasion as assessed by cell count, cell cycle, scatter, and transwell assays. AKT inhibition did not influence the cooperation on proliferation or invasion after HGF+EGF treatment, while ERK1/2 inhibition abolished MET/EGFR cooperation on proliferation. HGF+EGF treatment increased the duration of ERK1/2 and AKT activation compared to HGF or EGF alone. All these data indicate that a crosstalk between the EGF and HGF pathways in mammary epithelial cells may modulate the development of the mammary gland.

Activation of mitogen-activated protein kinases by a splice variant of GHRH receptor

Barabutis, N., Siejka, A., Schally, A. V, Block, N. L, Cai, R., Varga, J. L — Thu, 14 Jan 2010 17:51:21 PST

Hypothalamic GHRH controls the release of GH from the pituitary gland and also acts as a growth factor in a variety of cancers. The mitogenetic activity of GHRH is exerted through the binding to the pituitary type receptor (pGHRH-R) and its splice variants, mainly SV1. The intracellular signaling pathways that are activated upon the binding of GHRH to the SV1 receptor have not been elucidated. HeLa cervical cancer cells do not express GHRH or GHRH receptors (GHRHRs) and thus do not respond to GHRH or GHRH antagonists. In order to elucidate the mechanism of action of SV1 receptor, we transfected HeLa cells with plasmids for pcDNA3-GHRHR or pcDNA3-SV1. The transfected cells responded to both GHRH (1–29)NH₂ and GHRH antagonist MZ-5-156, as shown by an increase or decrease respectively in the proliferation rate in vitro and the expression of proliferative cell nuclear antigen. We also demonstrated that when the cells transfected with SV1 plasmid are stimulated with GHRH (1–29)NH₂, SV1 receptor activates the mitogen-activated protein kinases pathway (MAPKs), as shown previously for the cells that express pGHRH-R. Our results show, for the first time, the activation of the MAPKs cascade by the SV1 receptor. Since SV1 receptor is found in various tumors and mediates the responses to GHRH and synthetic antagonists, our findings shed light on the mechanism of action of SV1 receptor in cancer cells.

Rosiglitazone activation of PPAR{gamma} suppresses fractalkine signaling

Wan, Y., Evans, R. M — Thu, 14 Jan 2010 17:51:21 PST

The nuclear receptor peroxisome proliferator-activated receptor (PPAR) is a key transcriptional regulator of both lipid metabolism and inflammation. The importance of PPAR is accentuated by the widespread use of synthetic PPAR agonists, thiazolidinediones (such as rosiglitazone), as drugs for insulin resistance and type II diabetes. Fractalkine (FKN) and FKN receptor (FR) play an important role in the immune responses by regulating leukocyte migration and adhesion to inflamed peripheral tissues. In this study, we have identified a novel link between PPAR activation and FKN signaling. On one hand, the activation of PPAR by rosiglitazone in macrophages not only represses the transcription of the FR gene, but also prevents the plasma membrane translocation of the FR protein. On the other hand, the activation of PPAR by rosiglitazone in endothelial cells also impedes the nuclear export of FKN. Together, these data suggest that PPAR activation represses FKN signaling. These findings indicate a previously unrecognized mechanism that may contribute to the anti-inflammatory effect of PPAR.

Molecular aspects of thyroid hormone transporters, including MCT8, MCT10, and OATPs, and the effects of genetic variation in these transporters

van der Deure, W. M, Peeters, R. P, Visser, T. J — Thu, 17 Dec 2009 08:27:37 PST

Thyroid hormone is a pleiotropic hormone with widespread biological actions. For instance, adequate levels of thyroid hormone are critical for the development of different tissues such as the central nervous system, but are also essential for the regulation of metabolic processes throughout life. The biological activity of thyroid hormone depends not only on serum thyroid hormone levels, but is also regulated at the tissue level by the expression and activity of deiodinases, which activate thyroid hormone or mediate its degradation. In addition, thyroid hormone transporters are necessary for the uptake of thyroid hormone into target tissues. With the discovery of monocarboxylate transporter 8 (MCT8) as a specific thyroid hormone transporter and the finding that mutations in this transporter lead to a syndrome of severe psychomotor retardation and elevated serum 3,3',5-tri-iodothyronine levels known as the Allan–Herndon–Dudley syndrome, the interest in this area of research has greatly increased. In this review, we will focus on the molecular aspects of thyroid hormone transporters, including MCT8, MCT10, organic anion transporting polypeptides, and the effects of genetic variation in these transporters.

Cellular approaches to central nervous system remyelination stimulation: thyroid hormone to promote myelin repair via endogenous stem and precursor cells

Calza, L., Fernandez, M., Giardino, L. — Thu, 17 Dec 2009 08:27:37 PST

Brain and spinal cord repair is a very difficult task in view of the extremely limited repair capability of the mature central nervous system (CNS). Thus, cellular therapies are regarded as a new frontier for both acute and chronic neurological diseases characterized by neuron or oligodendroglia degeneration. Although cell replacement has been considered as the primary goal of such approaches, in recent years greater attention has been devoted to the possibility that new undifferentiated cells in damaged nervous tissue might also act in autocrine–paracrine fashion, regulating the micro-environment through the release of growth factor and cytokines, also regulating immune response and local inflammation. In this review, repair of demyelinating disease using endogenous cells will be discussed in view of the critical role played by thyroid hormones (THs) during developmental myelination, focusing on the following points: 1) endogenous stem and precursor cells during demyelinating diseases; 2) TH homeostasis in the CNS; 3) cellular and molecular mechanism regulated by TH during developmental myelination and 4) a working hypothesis to develop a rationale for the use of THs to improve remyelination through endogenous stem and precursor cells in the course of demyelinating diseases.

Maternal undernutrition increases pancreatic IGF-2 and partially suppresses the physiological wave of {beta}-cell apoptosis during the neonatal period

Thu, 17 Dec 2009 08:27:37 PST

Replication, neogenesis, and apoptosis play a main role in neonatal endocrine pancreas remodeling. IGFs are major contributors to β-cell growth and function and are highly sensitive to nutritional status. We previously showed that maternal malnutrition caused an increase in β-cell mass in fetuses related to the stimulation of β-cell proliferation due to increased pancreatic IGF-1. At 4 days of life, the β-cell mass was decreased in undernourished neonates and persisted until adult age. To clarify whether undernutrition disrupts islet remodeling, we quantified β-cell mass, neogenesis, replication, and apoptosis on days 4, 14, and 23. To determine the impact of food restriction on IGF ontogeny and the consequences for β-cell growth, we measured IGF-1/-2 protein content in pancreas and liver and pancreatic IGF-1 receptor (IGF-1R)-signaling pathway at the same days. Our results indicate that undernutrition alters the timing and intensity of neonatal β-cell ontogeny. However, although malnutrition causes β-cell deficiency in neonates, an active process of β-cell neogenesis and a lower incidence of β-cell apoptosis maintain the regenerative capacity of the endocrine pancreas. Interestingly, our data provide evidence that local production of IGFs seems to be instrumental in these processes. In particular, increased pancreatic IGF-2 in undernourished rats may contribute to the partial suppression of the developmental wave of β-cell apoptosis probably through the inhibition of glycogen synthase kinase-3. In addition, decreased pancreatic levels of IGFBP-1/-2/-3 in undernourished neonates could enhance IGF availability for interacting with IGF-1R/IR.

Stepwise loss of motilin and its specific receptor genes in rodents

He, J., Irwin, D. M, Chen, R., Zhang, Y.-P. — Thu, 17 Dec 2009 08:27:37 PST

Specific interactions among biomolecules drive virtually all cellular functions and underlie phenotypic complexity and diversity. Biomolecules are not isolated particles, but are elements of integrated interaction networks, and play their roles through specific interactions. Simultaneous emergence or loss of multiple interacting partners is unlikely. If one of the interacting partners is lost, then what are the evolutionary consequences for the retained partner? Taking advantages of the availability of the large number of mammalian genome sequences and knowledge of phylogenetic relationships of the species, we examined the evolutionary fate of the motilin (MLN) hormone gene, after the pseudogenization of its specific receptor, MLN receptor (MLNR), on the rodent lineage. We speculate that the MLNR gene became a pseudogene before the divergence of the squirrel and other rodents about 75 mya. The evolutionary consequences for the MLN gene were diverse. While an intact open reading frame for the MLN gene, which appears functional, was preserved in the kangaroo rat, the MLN gene became inactivated independently on the lineages leading to the guinea pig and the common ancestor of the mouse and rat. Gain and loss of specific interactions among biomolecules through the birth and death of genes for biomolecules point to a general evolutionary dynamic: gene birth and death are widespread phenomena in genome evolution, at the genetic level; thus, once mutations arise, a stepwise process of elaboration and optimization ensues, which gradually integrates and orders mutations into a coherent pattern.

Identification of protein kinases that control ovarian hormone release by selective siRNAs

Sirotkin, A. V, Ovcharenko, D., Mlyncek, M. — Thu, 17 Dec 2009 08:27:38 PST

The goal of this study was to identify protein kinases (PKs) that control the secretory activity of human ovarian cells. Cultured ovarian granulosa cells were transfected with 264 siRNA constructs that selectively block the expression of 88 known PKs. The efficiency of transfection and of silencing marker molecules (glyceraldehyde 3-phosphate dehydrogenase, GAPDH and CDC2/p34 PK) was validated by fluorescence microscopy, real-time reverse transcription-PCR, and immunocytochemistry. Release of steroid hormones (progesterone, P₄) and IGF1 was determined by RIA. siRNA suppressed the expression of marker molecules by up to 84%. P₄ release was suppressed after inhibiting 34 individual PKs and was stimulated after inhibiting 12 PKs. Blocking nine individual PKs inhibited IGF1 release, while the inactivation of 17 others stimulated IGF1 release. Together, these results demonstrate that the release of both steroid and peptide hormones by human ovarian cells is controlled by a large number of PKs, and that siRNA constructs may be useful tools for further defining the role of PKs in controlling ovarian secretory function.

Androgen-mediated improvement of body composition and muscle function involves a novel early transcriptional program including IGF1, mechano growth factor, and induction of {beta}-catenin

Thu, 17 Dec 2009 08:27:38 PST

Androgens promote anabolism in the musculoskeletal system while generally repressing adiposity, leading to lean body composition. Circulating androgens decline with age, contributing to frailty, osteoporosis, and obesity; however, the mechanisms by which androgens modulate body composition are largely unknown. Here, we demonstrate that aged castrated rats develop increased fat mass, reduced muscle mass and strength, and lower bone mass. Treatment with testosterone or 5-dihydrotestosterone (DHT) reverses the effects on muscle and adipose tissues while only aromatizable testosterone increased bone mass. During the first week, DHT transiently increased soleus muscle nuclear density and induced expression of IGF1 and its splice variant mechano growth factor (MGF) without early regulation of the myogenic factors MyoD, myogenin, monocyte nuclear factor, or myostatin. A genome-wide microarray screen was also performed to identify potential pro-myogenic genes that respond to androgen receptor activation in vivo within 24 h. Of 24 000 genes examined, 70 candidate genes were identified whose functions suggest initiation of remodeling and regeneration, including the type II muscle genes for myosin heavy chain type II and parvalbumin and the chemokine monocyte chemoattractant protein-1. Interestingly, Axin and Axin2, negative regulators of β-catenin, were repressed, indicating modulation of the β-catenin pathway. DHT increased total levels of β-catenin protein, which accumulated in nuclei in vivo. Likewise, treatment of C2C12 myoblasts with both IGF1Ea and MGF C-terminal peptide increased nuclear β-catenin in vitro. Thus, we propose that androgenic anabolism involves early downregulation of Axin and induction of IGF1, leading to nuclear accumulation of β-catenin, a pro-myogenic, anti-adipogenic stem cell regulatory factor.

Leu27IGF2 plays an opposite role to IGF1 to induce H9c2 cardiomyoblast cell apoptosis via G{alpha}q signaling

Thu, 19 Nov 2009 09:07:54 PST

This study examines the role of IGF2/mannose 6-phosphate receptor (IGF2R) signaling in the signaling transduction regulation and cell apoptosis in H9c2 cardiomyoblast cells. However, it is difficult to recognize the distinct activation of IGF2 signaling without interfacing with IGFI receptor (IGF1R) after exposure to IGF2. Leu27IGF2, an analog of IGF2 that interacts selectively with the IGF2R, was used to specifically activate IGF2R signaling in this study. DNA fragmentation and TUNEL assay revealed that in contrast to IGF1 treatment preventing angiotensin II and AG1024-induced cell apoptosis, Leu27IGF2 appears to synergistically increase apoptosis in those cells. We further found cell apoptosis induction and an increase in the active form of caspase 3 in the treatment of cells with Leu27IGF2, but not IGF1. To detect the interaction between IGF2R and Gq using the immunoprecipitation assay, we found that IGF2R could directly interact with Gq and after treatment with Leu27IGF2 the binding ability of Gq to IGF2R had increased. This sequentially resulted in the phosphorylation of phospholipase C-β, a key downstream modulator of Gq, on serine 537. Moreover, disruption of the Gq protein by small interferon RNA reduced the cell apoptosis induced by Leu27IGF2. Our findings demonstrate that IGF2R activation appears to induce cell apoptosis via Gq-deriving signaling cascades and its effect is completely different from IGF1R survival signaling.

Human aortic smooth muscle cells are insulin resistant at the receptor level but sensitive to IGF1 and IGF2

Chisalita, S I, Johansson, G S, Liefvendahl, E, Back, K, Arnqvist, H J — Thu, 19 Nov 2009 09:07:54 PST

Whether insulin, at physiological concentrations, has direct effects on vascular smooth muscle cells (VSMCs) remains controversial. Our aim was to characterize the mechanism for insulin resistance in VSMCs. For comparison, the effects of IGF1 and IGF2 were also studied. Cultured human aortic smooth muscle cells (HASMC) were used. Receptor mRNA was analyzed by quantitative reverse transcription PCR and receptor protein by ELISA and western blot. Biological effects were studied by thymidine incorporation and glucose accumulation. In HASMC, both mRNA and protein expression of IGF1 receptors (IGF1R) were fivefold higher compared to insulin receptor (IR). IR isoform A mRNA was 13-fold more expressed than IR isoform B. IR and IGF1R co-precipitated, indicating the presence of hybrid IR/IGF1R. Phosphorylation of the IGF1R β-subunit was obtained by IGF1 10^–9–10^–8 mol/l and IGF2 10^–8 mol/l. IR β-subunit was phosphorylated by IGF1 10^–8 mol/l but not by insulin. IGF1 stimulated IR substrate-1 and AKT at 10^–8 mol/l and extracellular signal-regulated kinases 1 and 2 at 10^–9–10^–8 mol/l respectively. IGF1 and 2 at a concentration of 10^–8–10^–7 mol/l significantly stimulated ³H-thymidine incorporation, whereas insulin did not. ¹⁴C-Glucose accumulation was stimulated by IGF1 or IGF2 10^–8–10^–7 mol/l, and also by insulin 10^–7 mol/l. Our results suggest that IGF1R and hybrid IR/IGF1R are activated by physiological concentrations of IGF1 and 2 in HASMC and this propagates downstream signaling and biological effects, while insulin has no effect on its receptor or downstream signaling probably due to a preponderance of IGF1R and incorporation of IR into hybrid IR/IGF1R.

Downregulation of peroxisome proliferator-activated receptor {alpha} and its coactivators in liver and skeletal muscle mediates the metabolic adaptations during lactation in mice

Gutgesell, A., Ringseis, R., Schmidt, E., Brandsch, C., Stangl, G. I, Eder, K. — Thu, 19 Nov 2009 09:07:54 PST

Previous studies have shown that genes involved in fatty acid uptake, fatty acid oxidation, and thermogenesis are downregulated in liver and skeletal muscle of rats during lactation. However, biochemical mechanisms underlying these important metabolic adaptations during lactation have not yet been elucidated. As all these genes are transcriptionally regulated by peroxisome proliferator-activated receptor (Ppar), we hypothesized that their downregulation is mediated by a suppression of Ppar during lactation. In order to investigate this hypothesis, we performed an experiment with lactating and nonlactating Ppar knockout and corresponding wild-type mice. In wild-type mice, lactation led to a considerable downregulation of Ppar, Ppar coactivators Pgc1 and Pgc1β, and Ppar target genes involved in fatty acid uptake, fatty acid oxidation, and thermogenesis in liver and skeletal muscle (P<0.05). Ppar knockout mice had generally a lower expression of all these Ppar target genes in liver and skeletal muscle. However, in those mice, lactation did not lower the expression of genes involved in fatty acid utilization and thermogenesis in liver and skeletal muscle. Expression levels of Ppar target genes in lactating wild-type mice were similar than in lactating or nonlactating Ppar knockout mice. In conclusion, the present findings suggest that downregulation of Ppar and its coactivators in tissues with high rates of fatty acid catabolism is responsible for the reduced utilization of fatty acids in liver and skeletal muscle and the reduced thermogenesis occurring in the lactating animal, which aim to conserve energy and metabolic substrates for milk production in the mammary gland.

Thioredoxin and thioredoxin reductase influence estrogen receptor {alpha}-mediated gene expression in human breast cancer cells

Rao, A. K, Ziegler, Y. S, McLeod, I. X, Yates, J. R, Nardulli, A. M — Thu, 19 Nov 2009 09:07:54 PST

Accumulation of reactive oxygen species (ROS) in cells damages resident proteins, lipids, and DNA. In order to overcome the oxidative stress that occurs with ROS accumulation, cells must balance free radical production with an increase in the level of antioxidant enzymes that convert free radicals to less harmful species. We identified two antioxidant enzymes, thioredoxin (Trx) and Trx reductase (TrxR), in a complex associated with the DNA-bound estrogen receptor (ER). Western analysis and immunocytochemistry were used to demonstrate that Trx and TrxR are expressed in the cytoplasm and in the nuclei of MCF-7 human breast cancer cells. More importantly, endogenously expressed ER, Trx, and TrxR interact and ER and TrxR associate with the native, estrogen-responsive pS2 and progesterone receptor genes in MCF-7 cells. RNA interference assays demonstrated that Trx and TrxR differentially influence estrogen-responsive gene expression and that together, 17β-estradiol, Trx, and TrxR alter hydrogen peroxide (H₂O₂) levels in MCF-7 cells. Our findings suggest that Trx and TrxR are multifunctional proteins that, in addition to modulating H₂O₂ levels and transcription factor activity, aid ER in regulating the expression of estrogen-responsive genes in target cells.

Surface translocation and tri-iodothyronine uptake of mutant MCT8 proteins are cell type-dependent

Kinne, A., Roth, S., Biebermann, H., Kohrle, J., Gruters, A., Schweizer, U. — Thu, 19 Nov 2009 09:07:54 PST

Mutations in the gene encoding the thyroid hormone transporter, monocarboxylate transporter 8 (MCT8), underlie severe mental retardation. We wanted to understand the functional consequences of a series of missense mutations in MCT8 in order to identify therapeutic options for affected patients. We established cell lines stably expressing 12 MCT8 variants in JEG1 and MDCK1 cells. The cell lines were characterized according to MCT8 mRNA and protein expression, tri-iodothyronine (T₃) transport activity, substrate K_M characteristics, surface expression, and responsiveness to T₃ preincubation and chemical chaperones. Functional activities of ins235V and L568P MCT8 mutants depend on the cell type in which they are expressed. These mutants and R271H exhibited considerable transport activity when present at the cell surface as verified by surface biotinylation and kinetic analysis. Most mutants, however, were inactive in T₃ transport even when present at the cell surface (e.g. S194F, A224V, F230, L512P). Preincubation of G558D with T₃ increased T₃ uptake in MDCK1 cells to a small, but significant, extent. Chemical chaperones were ineffective. The finding that the cell type determines surface expression and T₃ transport activities of missense mutants in MCT8 may be important to understand phenotypic variability among carriers of different mutations. In particular, the clinical observation that the severity of derangements of thyroid hormone levels does not correlate with mental impairments of the patients may be based on different residual activity of mutant MCT8 in different cell types.