Forkhead box protein O1 (FoxO1) regulates hepatic serine protease inhibitor B1 (serpinB1) expression in a non-cell-autonomous fashion.

FoxO proteins are major targets of insulin action, and FoxO1 mediates the effects of insulin on hepatic glucose metabolism. We reported previously that serpinB1 is a liver-secreted factor (hepatokine) that promotes adaptive ß-cell proliferation in response to insulin resistance in the liver-specific insulin receptor knockout (LIRKO) mouse. Here we report that FoxO1 plays a critical role in promoting serpinB1 expression in hepatic insulin resistance in a non-cell-autonomous manner. Mice lacking both the insulin receptor and FoxO1 (LIRFKO) exhibit reduced ß-cell mass compared with LIRKO mice because of attenuation of ß-cell proliferation. Although hepatic expression of serpinB1 mRNA and protein levels was increased in LIRKO mice, both the mRNA and protein levels returned to control levels in LIRFKO mice. Furthermore, liver-specific expression of constitutively active FoxO1 in transgenic mice induced an increase in hepatic serpinB1 mRNA and protein levels in refed mice. Conversely, serpinB1 mRNA and protein levels were reduced in mice lacking FoxO proteins in the liver. ChIP studies demonstrated that FoxO1 binds to three distinct sites located ∼9 kb upstream of the serpinb1 gene in primary mouse hepatocytes and that this binding is enhanced in hepatocytes from LIRKO mice. However, adenoviral expression of WT or constitutively active FoxO1 and insulin treatment are sufficient to regulate other FoxO1 target genes (IGFBP-1 and PEPCK) but not serpinB1 expression in mouse primary hepatocytes. These results indicate that liver FoxO1 promotes serpinB1 expression in hepatic insulin resistance and that non-cell-autonomous factors contribute to FoxO1-dependent effects on serpinB1 expression in the liver.

Coupling between Nutrient Availability and Thyroid Hormone Activation.

The activity of the thyroid gland is stimulated by food availability via leptin-induced thyrotropin-releasing hormone/thyroid-stimulating hormone expression. Here we show that food availability also stimulates thyroid hormone activation by accelerating the conversion of thyroxine to triiodothyronine via type 2 deiodinase in mouse skeletal muscle and in a cell model transitioning from 0.1 to 10% FBS. The underlying mechanism is transcriptional derepression of DIO2 through the mTORC2 pathway as defined in rictor knockdown cells. In cells kept in 0.1% FBS, there is DIO2 inhibition via FOXO1 binding to the DIO2 promoter. Repression of DIO2 by FOXO1 was confirmed using its specific inhibitor AS1842856 or adenoviral infection of constitutively active FOXO1. ChIP studies indicate that 4 h after 10% FBS-containing medium, FOXO1 binding markedly decreases, and the DIO2 promoter is activated. Studies in the insulin receptor FOXO1 KO mouse indicate that insulin is a key signaling molecule in this process. We conclude that FOXO1 represses DIO2 during fasting and that derepression occurs via nutritional activation of the PI3K-mTORC2-Akt pathway.

FoxO1 inhibits sterol regulatory element-binding protein-1c (SREBP-1c) gene expression via transcription factors Sp1 and SREBP-1c.

Induction of lipogenesis in response to insulin is critically dependent on the transcription factor, sterol regulatory element-binding protein-1c (SREBP-1c). FoxO1, a forkhead box class-O transcription factor, is an important mediator of insulin action, but its role in the regulation of lipid metabolism has not been clearly defined. We examined the effects of FoxO1 on srebp1 gene expression in vivo and in vitro. In vivo studies showed that constitutively active (CA) FoxO1 (CA-FoxO1) reduced basal expression of SREBP-1c mRNA in liver by ∼60% and blunted induction of SREBP-1c in response to feeding. In liver-specific FoxO knock-out mice, SREBP-1c expression was increased ∼2-fold. Similarly, in primary hepatocytes, CA-FoxO1 suppressed SREBP1-c expression and inhibited basal and insulin-induced SREBP-1c promoter activity. SREBP-1c gene expression is induced by the liver X receptor (LXR), but CA-FoxO1 did not block the activation of SREBP-1c by the LXR agonist TO9. Insulin stimulates SREBP-1c transcription through Sp1 and via "feed forward" regulation by newly synthesized SREBP-1c. CA-FoxO1 inhibited SREBP-1c by reducing the transactivational capacity of both Sp1 and SREBP-1c. In addition, chromatin immunoprecipitation assays indicate that FoxO1 can associate with the proximal promoter region of the srebp1 gene and disrupt the assembly of key components of the transcriptional complex of the SREBP-1c promoter. We conclude that FoxO1 inhibits SREBP-1c transcription via combined actions on multiple transcription factors and that this effect is exerted at least in part through reduced transcriptional activity of Sp1 and SREBP-1c and disrupted assembly of the transcriptional initiation complex on the SREBP-1c promoter.

The anti-neurodegenerative agent clioquinol regulates the transcription factor FOXO1a.

Many diseases of aging including AD (Alzheimer's disease) and T2D (Type 2 diabetes) are strongly associated with common risk factors, suggesting that there may be shared aging mechanisms underlying these diseases, with the scope to identify common cellular targets for therapy. In the present study we have examined the insulin-like signalling properties of an experimental AD 8-hydroxyquinoline drug known as CQ (clioquinol). The IIS [insulin/IGF-1 (insulin-like growth factor-1) signalling] kinase Akt/PKB (protein kinase B) inhibits the transcription factor FOXO1a (forkhead box O1a) by phosphorylating it on residues that trigger its exit from the nucleus. In HEK (human embryonic kidney)-293 cells, we found that CQ treatment induces similar responses. A key transcriptional response to IIS is the inhibition of hepatic gluconeogenic gene expression, and, in rat liver cells, CQ represses expression of the key gluconeogenic regulatory enzymes PEPCK (phosphoenolpyruvate carboxykinase) and G6Pase (glucose-6-phosphatase). The effects on FOXO1a and gluconeogenic gene expression require the presence of Zn2+ ions, reminiscent of much earlier studies examining diabetogenic properties of 8-hydroxyquinolines. Comparative investigation of the signalling properties of a panel of these compounds demonstrates that CQ alone exhibits FOXO1a regulation without diabetogenicity. Our results suggest that Zn2+-dependent regulation of FOXOs and gluconeogenesis may contribute to the therapeutic properties of this drug. Further investigation of this signalling response might illuminate novel pharmacological strategies for the treatment of age-related diseases.

Lactobacillus acidophilus Mitigates Osteoarthritis-Associated Pain, Cartilage Disintegration and Gut Microbiota Dysbiosis in an Experimental Murine OA Model.

To test probiotic therapy for osteoarthritis (OA), we administered Lactobacillus acidophilus (LA) by oral gavage (2×/week) after induction of OA by partial medial meniscectomy (PMM). Pain was assessed by von Frey filament and hot plate testing. Joint pathology and pain markers were comprehensively analyzed in knee joints, spinal cords, dorsal root ganglia and distal colon by Safranin O/fast green staining, immunofluorescence microscopy and RT-qPCR. LA acutely reduced inflammatory knee joint pain and prevented further OA progression. The therapeutic efficacy of LA was supported by a significant reduction of cartilage-degrading enzymes, pain markers and inflammatory factors in the tissues we examined. This finding suggests a likely clinical effect of LA on OA. The effect of LA treatment on the fecal microbiome was assessed by 16S rRNA gene amplicon sequencing analysis. LA significantly altered the fecal microbiota compared to vehicle-treated mice (PERMANOVA p < 0.009). Our pre-clinical OA animal model revealed significant OA disease modifying effects of LA as reflected by rapid joint pain reduction, cartilage protection, and reversal of dysbiosis. Our findings suggest that LA treatment has beneficial systemic effects that can potentially be developed as a safe OA disease-modifying drug (OADMD).

Hepatic FoxOs link insulin signaling with plasma lipoprotein metabolism through an apolipoprotein M/sphingosine-1-phosphate pathway.

Multiple beneficial cardiovascular effects of HDL depend on sphingosine-1-phosphate (S1P). S1P associates with HDL by binding to apolipoprotein M (ApoM). Insulin resistance is a major driver of dyslipidemia and cardiovascular risk. However, the mechanisms linking alterations in insulin signaling with plasma lipoprotein metabolism are incompletely understood. The insulin-repressible FoxO transcription factors mediate key effects of hepatic insulin action on glucose and lipoprotein metabolism. This work tested whether hepatic insulin signaling regulates HDL-S1P and aimed to identify the underlying molecular mechanisms. We report that insulin-resistant, nondiabetic individuals had decreased HDL-S1P levels, but no change in total plasma S1P. This also occurred in insulin-resistant db/db mice, which had low ApoM and a specific reduction of S1P in the HDL fraction, with no change in total plasma S1P levels. Using mice lacking hepatic FoxOs (L-FoxO1,3,4), we found that hepatic FoxOs were required for ApoM expression. Total plasma S1P levels were similar to those in controls, but S1P was nearly absent from HDL and was instead increased in the lipoprotein-depleted plasma fraction. This phenotype was restored to normal by rescuing ApoM in L-FoxO1,3,4 mice. Our findings show that insulin resistance in humans and mice is associated with decreased HDL-associated S1P. Our study shows that hepatic FoxO transcription factors are regulators of the ApoM/S1P pathway.

Hippocampus-dependent spatial learning and memory are impaired in growth hormone-deficient spontaneous dwarf rats.

Growth hormone (GH)/insulin-like growth factor-I deficiencies are known to cause alterations in brain development resulting in impairment of cognitive function. In order to investigate the behavioral phenotype of GH-deficient spontaneous dwarf rats (SDRs), we examined the behavior of the SDRs in the Morris water maze and Y-maze tasks. The SDRs showed severe deficits in spatial learning and memory compared to normal rats. The possibility that the cognitive impairment is associated with alteration of neurotransmitter systems was examined histologically following completion of the behavioral tests, using choline acetyltransferase (ChAT), vesicular glutamate transporter 1 (VGlut1) and glutamic acid decarboxylase (GAD6) immunohistochemistry as markers. In the SDRs the number of ChAT-stained basal forebrain cholinergic neurons was decreased. ChAT staining was also decreased in the hippocampus, one of the target areas of basal forebrain cholinergic neurons. Next, we examined the number of glutamatergic and GABAergic boutons in the hippocampal molecular layer and found a significant reduction in the density of VGlut1+ boutons and an increase in GAD6+ profiles, leading to a significantly reduced ratio in glutamatergic/GABAergic synapses. Finally, the number of newly generated cells in the subgranular zone of the hippocampus was significantly lower than in normal rats. Taken together, our data suggest that GH is an important regulator of hippocampus-dependent spatial learning and memory. The behavioral deficits in the SDRs may be explained by altered basal forebrain cholinergic innervation, imbalance in hippocampal glutamatergic/GABAergic synapses, and decreased neurogenesis in the hippocampus.

TORCs rev up gluconeogenesis.

Transducer of regulated CREB activity (TORC) proteins promote transactivation by the cAMP response element binding protein (CREB) and mediate effects of cAMP agonists on gene expression. Koo et al. now report that TORC phosphorylation and nuclear/cytoplasmic shuttling play a key role in the regulation of gluconeogenesis by cAMP. Control of TORC phosphorylation and function may integrate the effects of multiple factors involved in metabolic control, including cAMP agonists, insulin, and AMP kinases. TORCs, and kinases affecting TORC function, are promising new therapeutic targets for the treatment of diabetes mellitus.

FoxO1 and HNF-4 are involved in regulation of hepatic glucokinase gene expression by resveratrol.

Resveratrol, a polyphenol derived from grapes, exerts important effects on glucose and lipid metabolism, yet detailed mechanisms mediating these effects remain unknown. The liver plays a central role in energy homeostasis, and glucokinase (GK) is a key enzyme involved in glucose utilization. Resveratrol activates SIRT1 (sirtuin 1), which promotes deacetylation of the forkhead transcription factor FoxO1. Previously, we reported that FoxO1 can suppress and that HNF-4 can stimulate GK expression in the liver. Here, we examined the role of FoxO1 and HNF-4 in mediating resveratrol effects on liver GK expression. Resveratrol suppressed hepatic GK expression in vivo and in isolated hepatocytes, and knocking down FoxO1 with shRNAs disrupted this effect. Reporter gene, gel shift, supershift assay, and chromatin immunoprecipitation studies show that FoxO1 binds to the GK promoter and that the interplay between FoxO1 and HNF-4 within the GK promoter is essential for mediating the effects of resveratrol. Resveratrol promotes deacetylation of FoxO1 and enhances its recruitment to the FoxO-binding element. Conversely, resveratrol suppresses recruitment of HNF-4 to its binding site, and knockdown of FoxO1 blocks this effect of resveratrol. Coprecipitation and chromatin immunoprecipitation studies show that resveratrol enhances interaction between FoxO1 and HNF-4, reduces binding of HNF-4 to its own site, and promotes its recruitment to the FoxO site in a FoxO1-dependent manner. These results provide the first evidence that resveratrol represses GK expression via FoxO1 and that the interaction between FoxO1 and HNF-4 contributes to these effects of resveratrol.

FOXO transcription factors and VEGF neutralizing antibody enhance antiangiogenic effects of resveratrol.

Resveratrol (trans-3,5,4'-trihydroxystilbene), a compound found largely in the skins of red grapes and wines, possesses anti-cancer and anti-angiogenic properties and protects the cardiovascular system. However, the molecular mechanisms by which resveratrol inhibits angiogenesis are currently subjects of intense investigation. The purpose of this study was to examine whether FOXO transcription factors mediate anti-angiogenic effects of resveratrol, and whether vascular endothelial growth factor (VEGF) neutralizing antibody can enhance these effects of resveratrol. Inhibition of PI3 kinase (PI3K)/AKT and MEK/ERK pathways synergistically inhibited migration and capillary tube formation of Human Umbilical Vein Endothelial Cells (HUVECs) and further enhanced the anti-angiogenic effects of resveratrol. Inhibitors of AKT and MEK kinase synergistically inhibited cytoplasmic FOXO3a phosphorylation, which was accompanied by its nuclear translocation in HUVECs. Interestingly, inhibition of PI3K/AKT and MEK/ERK pathways synergistically induced FOXO transcriptional activity and inhibited cell migration and capillary tube formation. Antiangiogenic effects of resveratrol were enhanced by inhibitors of AKT and MEK. Phosphorylation-deficient mutants of FOXOs induced FOXO transcriptional activity, inhibited HUVEC cell migration, and capillary tube formation, and also enhanced antiangiogenic effects of resveratrol. Finally, VEGF neutralizing antibody enhanced the anti-proliferative and anti-angiogenic effects of resveratrol. In conclusion, regulation of FOXO transcription factors by resveratrol may play an important role in angiogenesis which is critical for cancer, diabetic retinopathy, rheumatoid arthritis, psoriasis, and cardiovascular disorders.

Identification of a periodontal pathogen and bihormonal cells in pancreatic islets of humans and a mouse model of periodontitis.

Results from epidemiological and prospective studies indicate a close association between periodontitis and diabetes. However the mechanisms by which periodontal pathogens influence the development of prediabetes/diabetes are not clear. We previously reported that oral administration of a periodontal pathogen, Porphyromonas gingivalis (Pg) to WT mice results in insulin resistance, hyperinsulinemia, and glucose intolerance and that Pg translocates to the pancreas. In the current study, we determined the specific localization of Pg in relation to mouse and human pancreatic α- and ß-cells using 3-D confocal and immunofluorescence microscopy and orthogonal analyses. Pg/gingipain is intra- or peri-nuclearly localized primarily in ß-cells in experimental mice and also in human post-mortem pancreatic samples. We also identified bihormonal cells in experimental mice as well as human pancreatic samples. A low percentage of bihormonal cells has intracellular Pg in both humans and experimental mice. Our data show that the number of Pg translocated to the pancreas correlates with the number of bihormonal cells in both mice and humans. Our findings suggest that Pg/gingipain translocates to pancreas, particularly ß-cells in both humans and mice, and this is strongly associated with emergence of bihormonal cells.

FoxO1 induces apoptosis in skeletal myotubes in a DNA-binding-dependent manner.

Recent studies indicate that FoxO transcription factors play an important role in promoting muscle atrophy. To study mechanisms mediating effects of FoxO proteins on muscle wasting, FoxO1-estrogen receptor fusion proteins that are activated by treatment with 4-hydroxytamoxifen (4-OH-T) were stably transfected in C(2)C(12) skeletal myoblasts using the pBABE retroviral system and grown into multinucleated skeletal myotubes. Activation of FoxO1 resulted in significant muscle atrophy, which was accompanied by DNA fragmentation, evidenced by terminal deoxynucleotidyl transferase dUTP-mediated nick end labeling. Cells expressing a DNA-binding-deficient form of FoxO1 also exhibited significant atrophy on FoxO1 activation but no hallmark signs of apoptosis. FoxO1 activation resulted in a significant increase in muscle atrophy F-box (MAFbx)/atrogin-1, muscle-specific RING finger protein 1 (MuRF-1), and Bcl-2-interacting mediator of cell death (Bim) gene expression, with no significant increase in Bcl-2/adenovirus E1B 19-kDa-interacting protein 3 (BNip3) gene expression. Although the ability of FoxO1 to induce MuRF-1 gene expression appeared to be independent of DNA binding, expression of MAFbx/atrogin-1 and Bim was significantly blunted in cells expressing DNA-binding-deficient FoxO1. BNip3 gene expression was significantly elevated in DNA-binding-deficient mutant cells. These findings indicate that FoxO1 promotes skeletal muscle atrophy through induction of proteolytic and apoptotic machinery via DNA-binding-dependent and -independent mechanisms.

Prolactin signaling through the short form of its receptor represses forkhead transcription factor FOXO3 and its target gene galt causing a severe ovarian defect.

Prolactin (PRL) is a hormone with over 300 biological activities. Although the signaling pathway downstream of the long form of its receptor (RL) has been well characterized, little is known about PRL actions upon activation of the short form (RS). Here, we show that mice expressing only RS exhibit an ovarian phenotype of accelerated follicular recruitment followed by massive follicular death leading to premature ovarian failure. Consequently, RS-expressing ovaries of young adults are depleted of functional follicles and formed mostly by interstitium. We also show that activation of RS represses the expression of the transcription factor Forkhead box O3 (FOXO3) and that of the enzyme galactose-1-phosphate uridyltransferase (Galt), two proteins known to be essential for normal follicular development. Our finding that FOXO3 regulates the expression of Galt and enhances its transcriptional activity indicates that it is the repression of FOXO3 by PRL acting through RS that prevents Galt expression in the ovary and causes follicular death. Coexpression of RL with RS prevents PRL inhibition of Galt, and the ovarian defect is no longer seen in RS transgenic mice that coexpress RL, suggesting that RL prevents RS-induced ovarian impairment. In summary, we show that PRL signals through RS and causes, in the absence of RL, a severe ovarian pathology by repressing the expression of FOXO3 and that of its target gene Galt. We also provide evidence of a link between the premature ovarian failure seen in mice expressing RS and in mice with FOXO3 gene deletion as well as in human with Galt mutation.

Associations of insulin resistance with cognition in individuals without diagnosed diabetes: Results from the Hispanic Community Health Study/Study of Latinos.

AIMS: Insulin resistance (IR) adversely impacts memory and executive functioning in non-Hispanic whites without diabetes. Less is known in Hispanics/Latinos, despite the fact that Hispanics/Latinos have higher rates of insulin resistance than non-Hispanic whites. We investigated the association between IR and cognition and its variation by age. METHODS: Data from 5987 participants 45-74 years old without diabetes from the Hispanic Community Health Study/Study of Latinos. IR was considered continuously using homeostasis model assessment for insulin resistance (HOMA-IR) and also dichotomized based on clinically relevant thresholds for hyperinsulinemia (fasting insulin > 84.73 pmol/L or HOMA-IR > 2.6) and sample-based norms (75th percentile of fasting insulin or HOMA-IR). Cognitive testing included the Brief Spanish English Verbal Learning Test (B-SEVLT), Verbal Fluency, and Digit Symbol Substitution. RESULTS: There was 90% overlap in participant categorization comparing clinically relevant and sample-based thresholds. In separate fully-adjusted linear regression models, age modified the association between HOMA-IR and Digit Symbol Substitution (p = 0.02); advancing age combined with higher HOMA-IR levels resulted in higher scores. Age also modified the association between clinically relevant hyperinsulinemia and B-SEVLT recall (p = 0.03); with increasing age came worse performance for individuals with hyperinsulinemia. CONCLUSION: The relationship of IR with cognition in Hispanics/Latinos without diabetes may reflect an age- and test-dependent state.

Forkhead box protein O1 negatively regulates skeletal myocyte differentiation through degradation of mammalian target of rapamycin pathway components.

The forkhead transcription factor forkhead box protein O1 (FoxO1), a downstream target of phosphatidylinositol 3-kinase/Akt signaling, has been reported to suppress skeletal myocyte differentiation, but the mechanism by which FoxO1 regulates myogenesis is not fully understood. We have previously demonstrated that a nutrient-sensing mammalian target of rapamycin (mTOR) pathway controls the autocrine production of IGF-II and the subsequent phosphatidylinositol 3-kinase/Akt signaling downstream of IGF-II in myogenesis. Here we report a regulatory loop connecting FoxO1 to the mTOR pathway. Inducible activation of a FoxO1 active mutant in the C2C12 mouse myoblasts blocks myogenic differentiation at an early stage and meanwhile leads to proteasome-dependent degradation of a specific subset of components in the mTOR signaling network, including mTOR, raptor, tuberous sclerosis complex 2, and S6 protein kinase 1. This function of FoxO1 requires new protein synthesis, consistent with the idea that a transcriptional target of FoxO1 may be responsible for the degradation of mTOR. We further show that active FoxO1 inhibits IGF-II expression at the transcriptional activation level, through the modulation of mTOR protein levels. Moreover, the addition of exogenous IGF-II fully rescues myocyte differentiation from FoxO inhibition. Taken together, we propose that the mTOR-IGF-II pathway is a major mediator of FoxO's inhibitory function in skeletal myogenesis.

Regulation of SREBP1c gene expression in skeletal muscle: role of retinoid X receptor/liver X receptor and forkhead-O1 transcription factor.

Sterol regulatory element binding protein 1c (SREBP1c) is a master regulator of lipogenic gene expression in liver and adipose tissue, where its expression is regulated by a heterodimer of nuclear receptor-type transcription factors retinoid X receptor-alpha (RXRalpha) and liver X receptor-alpha (LXRalpha). Despite the potential importance of SREBP1c in skeletal muscle, little is known about the regulation of SREBP1c in that setting. Here we report that gene expression of RXRgamma is markedly decreased by fasting and is restored by refeeding in mouse skeletal muscle, in parallel with changes in gene expression of SREBP1c. RXRgamma or RXRalpha, together with LXRalpha, activate the SREBP1c promoter in vitro. Moreover, transgenic mice overexpressing RXRgamma specifically in skeletal muscle showed increased gene expression of SREBP1c with increased triglyceride content in their skeletal muscles. In contrast, transgenic mice overexpressing the dominant-negative form of RXRgamma showed decreased SREBP1c gene expression. The expression of Forkhead-O1 transcription factor (FOXO1), which can suppress the function of multiple nuclear receptors, is negatively correlated to that of SREBP1c in skeletal muscle during nutritional change. Moreover, transgenic mice overexpressing FOXO1 specifically in skeletal muscle exhibited decreased gene expression of both RXRgamma and SREBP1c. In addition, FOXO1 suppressed RXRalpha/LXRalpha-mediated SREBP1c promoter activity in vitro. These findings provide in vivo and in vitro evidence that RXR/LXR up-regulates SREBP1c gene expression and that FOXO1 antagonizes this effect of RXR/LXR in skeletal muscle.

Disruption of growth hormone signaling retards prostate carcinogenesis in the Probasin/TAg rat.

We asked whether down-regulation of GH signaling could block carcinogenesis in the Probasin/TAg rat, a model of aggressive prostate cancer. The Spontaneous Dwarf rat, which lacks GH due to a mutation (dr) in its GH gene, was crossed with the Probasin/TAg rat, which develops prostate carcinomas at 100% incidence by 15 wk of age. Progeny were heterozygous for the TAg oncogene and homozygous for either the wild-type GH gene (TAg/Gh(+/+)) or the dr mutation (TAg/Gh(dr/dr)). Prostate tumor incidence and burden were significantly reduced, and tumor latency was delayed in TAg/Gh(dr/dr) rats relative to TAg/Gh(+/+) controls. At 25 wk of age, loss of GH resulted in a 20 and 80% decrease in the area of microinvasive carcinoma in the dorsal and lateral lobes, respectively. By 52 wk of age, invasive prostate adenocarcinomas were observed in all TAg/Gh(+/+) rats, whereas the majority of TAg/Gh(dr/dr) did not develop invasive tumors. Suppression of carcinogenesis could not be attributed to alterations in prostate expression of TAg or androgen receptor or changes in serum testosterone levels. As carcinogenesis progressed in TAg/Gh(+/+) rats, prostate GHR mRNA and protein expression increased significantly, but prostate IGF-I receptor mRNA and protein levels dropped. Furthermore, serum IGF-I and prostate IGF-I levels did not change significantly over the course of carcinogenesis. These findings suggest that GH plays a dominant role in progression from latent to malignant prostate cancer driven by the powerful probasin/TAg fusion gene in rats and suggest that GH antagonists may be effective at treating human prostate cancer.

Signal transducer and activator of transcription (Stat) 5b-mediated inhibition of insulin-like growth factor binding protein-1 gene transcription: a mechanism for repression of gene expression by growth hormone.

GH plays a central role in controlling somatic growth, tissue regeneration, and intermediary metabolism in most vertebrate species through mechanisms dependent on the regulation of gene expression. Recent studies using transcript profiling have identified large cohorts of genes whose expression is induced by GH. Other results have demonstrated that signal transducer and activator of transcription (Stat) 5b, a latent transcription factor activated by the GH receptor-associated protein kinase, Jak2, is a key agent in the GH-stimulated gene activation that leads to somatic growth. By contrast, little is known about the steps through which GH-initiated signaling pathways reduce gene expression. Here we show that Stat5b plays a critical role in the GH-regulated inhibition of IGF binding protein-1 gene transcription by impairing the actions of the FoxO1 transcription factor on the IGF binding protein-1 promoter. Additional observations using transcript profiling in the liver indicate that Stat5b may be a general mediator of GH-initiated gene repression. Our results provide a model for understanding how GH may simultaneously stimulate and inhibit the expression of different cohorts of genes via the same transcription factor, potentially explaining how GH action leads to integrated biological responses in the whole organism.

Transcriptional cross talk between the forkhead transcription factor forkhead box O1A and the progesterone receptor coordinates cell cycle regulation and differentiation in human endometrial stromal cells.

Differentiation of human endometrial stromal cells (HESCs) into decidual cells is associated with induction of the forkhead transcription factor forkhead box O1A (FOXO1). We performed a genomic screen to identify decidua-specific genes under FOXO1 control. Primary HESCs were transfected with small interfering RNA targeting FOXO1 or with nontargeting control small interfering RNA before treatment with a cAMP analogue and the progestin, medroxyprogesterone acetate for 72 h. Total RNA was processed for whole genome analysis using high-density oligonucleotide arrays. We identified 3405 significantly regulated genes upon decidualization of HESCs, 507 (15.3%) of which were aberrantly expressed upon FOXO1 knockdown. Among the most up-regulated FOXO1-dependent transcriptional targets were WNT signaling-related genes (WNT4, WNT16 ), the insulin receptor (INSR), differentiation markers (PRL, IGFBP1, and LEFTY2), and the cyclin-dependent kinase inhibitor p57(Kip2) (CDKN1C). Analysis of FOXO1-dependent down-regulated genes uncovered several factors involved in cell cycle regulation, including CCNB1, CCNB2, MCM5, CDC2 and NEK2. Cell viability assay and cell cycle analysis demonstrated that FOXO1 silencing promotes proliferation of differentiating HESCs. Using a glutathione-S-transferase pull-down assay, we confirmed that FOXO1 interacts with progesterone receptor, irrespectively of the presence of ligand. In agreement, knockdown of PR disrupted the regulation of FOXO1 target genes involved in differentiation (IGFBP1, PRL, and WNT4) and cell cycle regulation (CDKN1, CCNB2 and CDC2) in HESCs treated with either cAMP plus medroxyprogesterone acetate or with cAMP alone. Together, the data demonstrate that FOXO1 engages in transcriptional cross talk with progesterone receptor to coordinate cell cycle regulation and differentiation of HESCs.

Effect of periodontitis on insulin resistance and the onset of type 2 diabetes mellitus in Zucker diabetic fatty rats.

BACKGROUND: Studies indicate that an association exists between periodontitis and type 2 diabetes mellitus (T2DM) and/or obesity, with chronic inflammation hypothesized as the common denominator. The purpose of this study was to determine the causal effect of periodontitis and the concomitant impact of diet on the onset of insulin resistance (IR) and T2DM using a rat model system that simulates human obesity and T2DM. METHODS: Twenty-eight, 5-week-old female Zucker diabetic fatty (ZDF, fa/fa) rats were divided into four groups of seven animals: high-fat fed-periodontitis (HF/P), high-fat fed-no periodontitis (HF/C), low-fat fed-periodontitis (LF/P), and low-fat fed-no periodontitis (LF/C). Periodontitis was induced by ligature placement. Fasting plasma insulin and glucose levels were measured, and glucose tolerance tests were performed to assess glucose homeostasis, IR, and the onset of T2DM. The level of tumor necrosis factor-alpha (TNF-alpha), leptin, triglycerides, and free fatty acids were determined in week 13 at sacrifice. RESULTS: HF/P rats developed more severe IR compared to HF/C rats (P <0.01) and LF/P or LF/C rats (P <0.001) as measured by fasting insulin levels and homeostasis model assessment analysis. The onset of severe IR occurred approximately 3 weeks earlier in HF/P rats compared to HF/C rats. HF/P rats developed impaired (110 to 125 mg/dl) and frank fasting hyperglycemia (>125 mg/dl) 2 weeks earlier than HF/C rats. There was no difference in the severity and onset of IR and T2DM between LF/P and LF/C rats. The level of TNF-alpha was significantly higher in HF/P rats compared to HF/C rats (P <0.01). CONCLUSION: Periodontitis accelerated the onset of severe IR and impaired glucose homeostasis in high-fat fed ZDF rats.