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Background: This study aimed to identify the roles of L-tryptophan (Trp) and its rate-limiting enzymes on the receptivity of bovine endometrial epithelial cells. Real-time PCR was conducted to analyze the differential expression of genes between different groups of bovine endometrial epithelial cells. Western blot was performed to detect Cyclooxygenase-2 (COX2) expression after treatment with Trp or kynurenine (the main metabolites of Trp). The kynurenine assay was used to examine if Trp or prostaglandin E2 (PGE2) can increase the production of kynurenine in the bovine endometrial epithelial cells., Results: Trp significantly stimulates insulin growth factor binding protein 1 (IGFBP1) expression, a common endometrial marker of conceptus elongation and uterus receptivity for ruminants. When bovine endometrial epithelial cells are treated with Trp, tryptophan hydroxylase-1 remains unchanged, but tryptophan 2,3-dioxygenase 2 (TDO2) is significantly increased, suggesting tryptophan is mainly metabolized through the kynurenine pathway. Kynurenine significantly stimulates IGFBP1 expression. Furthermore, Trp and kynurenine significantly increase the expression of aryl hydrocarbon receptor (AHR). CH223191, an AHR inhibitor, abrogates the induction of Trp and kynurenine on IGFBP1. PGE2 significantly induces the expression of TDO2, AHR, and IGFBP1., Conclusions: The regulation between Trp / kynurenine and PGE2 may be crucial for the receptivity of the bovine uterus., (© 2024. The Author(s).)

Backgrounds: Nonalcoholic fatty liver disease (NAFLD) exhibits a close association with osteoporosis. This work aims to assess the potential effects of NAFLD on the progression of osteopenia in animal models., Methods: Forty-eight C57BL/6 female mice were randomly divided to wild-type (WT) group and high-fat diet (HFD) group. The corresponding detections were performed after sacrifice at 16, 24 and 32 weeks, respectively., Results: At 16 weeks, an remarkable increase in body weight and lipid aggregation in the hepatocytes of HFD group was observed compared to the WT group, while the bone structure parameters showed no significant difference. At 24 weeks, the levels of TNF-α and IL-6 in NAFLD mice were significantly increased, while the level of osteoprotegerin mRNA in bone tissue was decreased, and the level of receptor activator of nuclear factor Kappa-B ligand mRNA was increased. Meanwhile, the function of osteoclasts was increased, and the bone microstructure parameters showed significant changes. At 32 weeks, in the HFD mice, the mRNA levels of insulin-like growth factor-1 (IGF-1), runt-related transcription factor 2, and osterix mRNA were reduced, while the insulin-like growth factor binding protein-1 (IGFBP-1) level was increased. Simultaneously, the osteoblast function was decreased, and the differences of bone structure parameters were more significant, showing obvious osteoporosis., Conclusions: The bone loss in HFD mice is pronounced as NAFLD progresses, and the changes of the TNF-α, IL-6, IGF-1, and IGFBP-1 levels may play critical roles at the different stages of NAFLD in HFD., (© 2023. The Author(s).)

Background: The progress of treatments of childhood acute lymphoblastic leukemia (ALL) has made it possible to reach a survival rate superior to 80%. However, the treatments lead to several long-term adverse effects, including cardiac toxicity. Although studies have reported associations between genetic variants and cardiorespiratory fitness, none has been performed on childhood ALL survivors., Methods: We performed whole-exome sequencing in 239 childhood ALL survivors from the PETALE cohort. Germline variants (both common and rare) in selected set of genes (N = 238) were analyzed for an association with cardiorespiratory fitness., Results: Our results showed that the common variant in the TTN gene was significantly associated with a low cardiorespiratory fitness level (p = 0.0005) and that the LEPR, IGFBPI and ENO3 genes were significantly associated with a low cardiorespiratory fitness level in female survivors (p ≤ 0.002). Also, we detected an association between the low cardiorespiratory fitness level in participants that were stratified to the "high risk" prognostic group and functionally predicted rare variants in the SLC22A16 gene (p = 0.001). Positive associations between cardiorespiratory fitness level and trainability genes were mainly observed in females., Conclusions: For the first time, we observed that low cardiorespiratory fitness in childhood ALL survivors can be associated with variants in genes related to subjects' trainability. These findings could allow better childhood ALL patient follow-up tailored to their genetic profile and cardiorespiratory fitness, which could help reduce at least some of the burden of long-term adverse effects of treatments.

Background: During decidualization in endometrial stromal cells (ESCs), expressions of a number of genes and epigenetic modifications of histones are altered. However, there is little information about whether DNA methylation, which is another epigenetic mechanism, also changes during decidualization. Here, we examined the genome-wide DNA methylation profiles in ESCs during decidualization and their associations with the changes of gene expressions and histone modifications., Results: ESCs were incubated with estradiol and medroxyprogesterone acetate for 14 days to induce decidualization. The genome-wide DNA methylation profiles were compared between the non-decidualized ESCs and the decidualized ESCs. Of 482,005 CpGs, only 23 CpGs (0.0048%) showed different DNA methylation statuses. The DNA methylation statuses of the differentially expressed genes and the regions with different histone modifications (H3K4 tri-methylation and H3K27 acetylation) were also compared between the ESCs. In the upregulated and downregulated genes in decidualized ESCs, DNA methylation statuses around the promoter region of the genes did not significantly differ between the ESCs. In the regions with different histone modification, DNA methylation statuses did not differ between the ESCs. The differentially expressed genes and the differential histone modification regions were hypomethylated., Conclusions: Culturing ESCs with estrogen/progesterone did not distort the physiological pattern of DNA methylation, although mRNA expression and histone modifications were dynamically altered. A genome-wide DNA methylation analysis revealed stable DNA methylation statuses during decidualization in human endometrial stromal cells. DNA hypomethylation is maintained for the variable changes of histone modifications and gene expression during decidualization.

Background: The contribution of genetic polymorphisms to the large inter-individual variation in mammographic density (MD) changes following starting and stopping use of estrogen and progestin combined therapy (EPT) has not been well-studied. Previous studies have shown that circulating levels of insulin-like growth factors are associated with MD and cross-talk between estrogen signaling and growth factors is necessary for cell proliferation in the breast. We evaluated single nucleotide polymorphisms (SNPs) in growth factor genes in association with MD changes after women stop EPT use., Methods: We genotyped 191 SNPs in 13 growth factor pathway genes in 284 non-Hispanic white California Teachers Study participants who previously used EPT and collected their mammograms before and after quitting EPT. Percent MD was assessed using a computer-assisted method. Change in percent MD was calculated by subtracting percent MD of an 'off-EPT' mammogram from percent MD of an 'on-EPT' (i.e. baseline) mammogram. We used multivariable linear regression analysis to investigate the association between SNPs and change in percent MD. We calculated P-values corrected for multiple testing within a gene (P adj )., Results: Rs1983210 in INHA and rs35539615 in IGFBP1/3 showed the strongest associations. Per minor allele of rs1983210, the absolute change in percent MD after stopping EPT use decreased by 1.80% (a difference in absolute change in percent MD) (P adj = 0.021). For rs35539615, change in percent MD increased by 1.79% per minor allele (P adj = 0.042). However, after applying a Bonferroni correction for the number of genes tested, these associations were no longer statistically significant., Conclusions: Genetic variation in growth factor pathway genes INHA and IGFBP1/3 may predict longitudinal MD change after women quit EPT. The observed differences in EPT-associated changes in percent MD in association with these genetic polymorphisms are modest but may be clinically significant considering that the magnitude of absolute increase in percent MD reported from large clinical trials of EPT ranged from 3% to 7%.

The insulin-like growth factor-2 mRNA-binding protein 1 (IGF2BP1) plays essential roles in embryogenesis and carcinogenesis. IGF2BP1 serves as a post-transcriptional fine-tuner regulating the expression of some essential mRNA targets required for the control of tumor cell proliferation and growth, invasion, and chemo-resistance, associating with a poor overall survival and metastasis in various types of human cancers. Therefore, IGF2BP1 has been traditionally regarded as an oncogene and potential therapeutic target for cancers. Nevertheless, a few studies have also demonstrated its tumor-suppressive role. However, the details about the contradictory functions of IGF2BP1 are unclear. The growing numbers of microRNAs (miRNAs) and long non-coding RNAs (lncRNAs) have been identified as its direct regulators, during tumor cell proliferation, growth, and invasion in multiple cancers. Thus, the mechanisms of post-transcriptional modulation of gene expression mediated by IGF2BP1, miRNAs, and lncRNAs in determining the fate of the development of tissues and organs, as well as tumorigenesis, need to be elucidated. In this review, we summarized the tissue distribution, expression, and roles of IGF2BP1 in embryogenesis and tumorigenesis, and focused on modulation of the interconnectivity between IGF2BP1 and its targeted mRNAs or non-coding RNAs (ncRNAs). The potential use of inhibitors of IGF2BP1 and its related pathways in cancer therapy was also discussed.

Background: Wharton's jelly-derived mesenchymal stem cells (WJ-MSCs) are a novel and promising strategy for tissue engineering because of their ability to differentiate into many cell types. We characterized the differentiation of WJ-MSCs into endometrial epithelial cell (EEC)-like and endometrial stromal cell (ESC)-like cells and assessed the effect of 17β-estradiol and 8-Br-cAMP on the differentiation system., Methods: WJ-MSCs were treated in two ways to differentiate into EEC-like and ESC-like cells respectively: cocultured with ESCs in control/differentiation medium (17β-estradiol, growth factors); and cultured in control/differentiation medium (8-Br-cAMP alone or 8-Br-cAMP plus 17β-estrogen and growth factors). Three signaling pathway inhibitors (SB203580, PD98059, H89) were used to investigate the mechanism of WJ-MSC differentiation into ESC-like cells. Immunofluorescence, western blot and flow cytometry analyses were used to analyze expression of epithelial markers and stromal cell markers. Enzyme-linked immunosorbent assays were used to test the production of secretory proteins associated with the differentiation of ESC-like cells., Results: 17β-estradiol at 1 μM downregulated vimentin and CD13 and upregulated cytokeratin and CD9 proteins, promoting the differentiation of WJ-MSCs into EEC-like cells in the coculture system. 8-Br-cAMP at 0.5 mM upregulated vimentin and CD13 and downregulated CK and CD9, promoting the differentiation of WJ-MSCs into ESC-like cells. Prolactin (PRL) and insulin-like growth factor-binding protein 1 (IGFBP1) were upregulated and the protein kinase A (PKA) signaling pathway was activated, whereas extracellular signal-regulated (ERK)1/2 and p38 mitogen-activated protein kinase (MAPK) were not affected., Conclusions: 17β-estradiol at 1 μM is a good inducer for facilitating the differentiation of WJ-MSCs into EEC-like cells. 8-Br-cAMP plus estrogen and growth factors can induce the differentiation of WJ-MSCs into ESC-like cells. During the differentiation of WJ-MSCs into ESC-like cells, PRL and IGFBP1 were upregulated by the treatment and the PKA signaling pathway was activated, whereas ERK1/2 and p38 MAPK were not affected. These findings suggest a promising approach to the treatment of endometrial damage and other endometrial diseases and suggest new applications for WJ-MSCs in clinical practice.

Background: Chronic endometritis (CE) is a continuous inflammation of uterine endometrium, and it is usually symptomless. As CE has been thought not to affect the reproductive status and general health of affected women, its significance has not been explored. However, recent studies have shown that CE is related with repeated implantation failures after in vitro fertilization-embryo transfer, unexplained infertility, and recurrent miscarriages. As decidua differentiates to support the implantation process and maintains the pregnancy, we hypothesized that CE may influence the process of decidualization., Methods: Seventeen patients were employed in the experiment involving culture of endometrial stromal cells (ESCs). After obtaining endometrial samples, ESCs were harvested and cultured for 13 days. The concentrations in culture media and the protein expressions in ESCs of prolactin (PRL) and insulin-like growth factor binding protein-1 (IGFBP-1), two well known decidualization markers used in a large number of in vitro models, were analyzed by ELISA and Western blotting, respectively, and the cell numbers were also counted. The mRNA levels of PRL and IGFBP-1 were tested by quantitative real time polymerase chain reaction (RT-PCR). Since sex hormone induce proliferation and differentiation to decidua via binding to the sex hormone receptors (ERα, ERβ, PRA, and PRB), their expression was assessed in another 17 patients' paraffin-embedded endometrial tissue specimens by immunohistochemistry and semi-quantified by H-score., Results: Increased cell numbers and reduced secretion of PRL and IGFBP-1 were detected by ELISA in the ESCs of CE patients after culture for 13 days compared with non-CE patients. The decreased protein expression of IGFBP-1 in ESCs of CE patients was detected by Western blotting. The decreased expression of PRL mRNA and IGFBP-1 mRNA were detected by RT-PCR. Increased expressions of ERα, ERβ, PRA, and PRB were observed in the stromal cells of CE patients in comparison to non-CE patients, whereas increased expressions of ERα and ERβ were detected in the glandular cells of CE., Conclusion: Our data suggests that CE modifies decidualization of human ESC through untuning the function of sex steroid hormone receptor.

Background: Ursolic acid (UA), a natural pentacyclic triterpenoid, exerts anti-tumor effects in various cancer types including hepatocellular carcinoma (HCC). However, the molecular mechanisms underlying this remain largely unknown., Methods: Cell viability and cell cycle were examined by MTT and Flow cytometry assays. Western blot analysis was performed to measure the phosphorylation and protein expression of p38 MAPK, insulin-like growth factor (IGF) binding protein 1 (IGFBP1) and forkhead box O3A (FOXO3a). Quantitative real-time PCR (qRT-PCR) was used to examine the mRNA levels of IGFBP1 gene. Small interfering RNAs (siRNAs) method was used to knockdown IGFBP1 gene. Exogenous expressions of IGFBP1 and FOXO3a were carried out by transient transfection assays. IGFBP1 promoter activity was measured by Secrete-Pair™ Dual Luminescence Assay Kit . In vivo nude mice xenograft model and bioluminescent imaging system were used to confirm the findings in vitro., Results: We showed that UA stimulated phosphorylation of p38 MAPK. In addition, UA increased the protein, mRNA levels, and promoter activity of IGFBP1, which was abrogated by the specific inhibitor of p38 MAPK (SB203580). Intriguingly, we showed that UA increased the expression of FOXO3a and that overexpressed FOXO3a enhanced phosphorylation of p38 MAPK, all of which were not observed in cells silencing of endogenous IGFBP1 gene. Moreover, exogenous expressed IGFBP1 strengthened UA-induced phosphorylation of p38 MAPK and FOXO3a protein expression, and more importantly, restored the effect of UA-inhibited growth in cells silencing of endogenous IGFBP1 gene. Consistent with these, UA suppressed tumor growth and increased phosphorylation of p38 MAPK, protein expressions of IGFBP1 and FOXO3a in vivo., Conclusion: Collectively, our results show that UA inhibits growth of HCC cells through p38 MAPK-mediated induction of IGFBP1 and FOXO3a expression. The interactions between IGFBP1 and FOXO3a, and feedback regulatory loop of p38 MAPK by IGFBP1 and FOXO3a resulting in reciprocal pathways, contribute to the overall effects of UA. This in vitro and in vivo study corroborates a potential novel mechanism by which UA controls HCC growth and implies that the rational targeting IGFBP1 and FOXO3a can be potential for the therapeutic strategy against HCC.

Background: Hedgehog signaling plays an important role in embryonic development, organogenesis and cancer. In the adult liver, Hedgehog signaling in non-parenchymal cells has been found to play a role in certain disease states such as fibrosis and cirrhosis. However, whether the Hedgehog pathway is active in mature healthy hepatocytes and is of significance to liver function are controversial., Findings: Two types of mice with distinct conditional hepatic deletion of the Smoothened gene, an essential co-receptor protein of the Hedgehog pathway, were generated for investigating the role of Hedgehog signaling in mature hepatocytes. The knockout animals (KO) were inconspicuous and healthy with no changes in serum transaminases, but showed a slower weight gain. The liver was smaller, but presented a normal architecture and cellular composition. By quantitative RT-PCR the downregulation of the expression of Indian hedgehog (Ihh) and the Gli3 transcription factor could be demonstrated in healthy mature hepatocytes from these mice, whereas Patched1 was upregulated. Strong alterations in gene expression were also observed for the IGF axis. While expression of Igf1 was downregulated, that of Igfbp1 was upregulated in the livers of both genders. Corresponding changes in the serum levels of both proteins could be detected by ELISA. By activating and inhibiting the transcriptional output of Hedgehog signaling in cultured hepatocytes through siRNAs against Ptch1 and Gli3, respectively, in combination with a ChIP assay evidence was collected indicating that Igf1 expression is directly dependent on the activator function of Gli3. In contrast, the mRNA level of Igfbp1 appears to be controlled through the repressor function of Gli3, while that of Igfbp2 and Igfbp3 did not change. Interestingly, body weight of the transgenic mice correlated well with IGF-I levels in both genders and also with IGFBP-1 levels in females, whereas it did not correlate with serum growth hormone levels., Conclusions: Our results demonstrate for the first time that Hedgehog signaling is active in healthy mature mouse hepatocytes and that it has considerable importance for IGF-I homeostasis in the circulation. These findings may have various implications for mouse physiology including the regulation of body weight and size, glucose homeostasis and reproductive capacity.

Background: Alcohol consumption is a major cause of liver disease in humans. The use and monitoring of biomarkers associated with early, pre-clinical stages of alcohol-induced liver disease (pre-ALD) could facilitate diagnosis and treatment, leading to improved outcomes., Methods: We investigated the pathological, transcriptomic and protein changes in early stages of pre-ALD in mice fed the Lieber-Decarli liquid diet with or without alcohol for four months to identify biomarkers for the early stage of alcohol induced liver injury. Mice were sampled after 1, 2 and 4 months treatment., Results: Pathological examination revealed a modest increase in fatty liver changes in alcohol-treated mice. Transcriptomics revealed gene alterations at all time points. Most notably, the Igfbp1 (Insulin-Like Growth Factor Binding Protein 1) was selected as the best candidate gene for early detection of liver damage since it showed early and continuously enhanced induction during the treatment course. Consistent with the microarray data, both Igfbp1mRNA expression in the liver tissue and the IGFBP1 serum protein levels showed progressive and significant increases over the course of pre-ALD development., Conclusions: The results suggest that in conjunction with other tests, serum IGFBPI protein could provide an easily measured biomarker for early detection of alcohol-induced liver injury in humans.

Background: Early life body size and circulating levels of IGF-1 and IGFBP-3 have been linked to increased risks of breast and other cancers, but it is unclear whether these exposures act through a common mechanism. Previous studies have examined the role of IGF-1 and IGFBP-3 genetic variation in relation to adult height and body size, but few studies have examined associations with birthweight and childhood size., Methods: We examined whether htSNPs in IGF-1 and the IGFBP-1/IGFBP-3 gene region are associated with the self-reported outcomes of birthweight, body fatness at ages 5 and 10, and body mass index (BMI) at age 18 among healthy women from the Nurses' Health Study (NHS) and NHSII. We used ordinal logistic regression to model odds ratios (ORs) and 95% confidence intervals (CI) of a one category increase for birthweight and somatotypes at ages 5 and 10. We used linear regression to model associations with BMI at age 18., Results: Among 4567 healthy women in NHS and NHSII, we observed no association between common IGF-1 or IGFBP-1/IGFBP-3 SNPs and birthweight, body fatness at ages 5 and 10, or BMI at age 18., Conclusions: Common IGF-1 and IGFBP-1/IGFBP-3 SNPs are not associated with body size in early life.

Background: Previously we performed a linkage scan of 638 African American affected sibling pairs (ASP) with type 2 diabetes (T2D) enriched for end-stage renal disease (ESRD). Ordered subset linkage analysis (OSA) revealed a linkage peak on chromosome 7p in the subset of families with earlier age of T2D diagnosis., Methods: We fine mapped this region by genotyping 11 additional polymorphic markers in the same ASP and investigated a total of 68 single nucleotide polymorphisms (SNPs) in functional candidate genes (GCK1, IL6, IGFBP1 and IGFBP3) for association with age of T2D diagnosis, age of ESRD diagnosis, duration of T2D to onset of ESRD, body mass index (BMI) in African American cases and T2D-ESRD in an African American case-control cohort. OSA of fine mapping markers supported linkage at 28 cM on 7p (near D7S3051) in early-onset T2D families (max. LOD = 3.61, P = 0.002). SNPs in candidate genes and 70 ancestry-informative markers (AIMs) were evaluated in 577 African American T2D-ESRD cases and 596 African American controls., Results: The most significant association was observed between ESRD age of diagnosis and SNP rs730497, located in intron 1 of the GCK1 gene (recessive T2D age-adjusted P = 0.0006). Nominal associations were observed with GCK1 SNPs and T2D age of diagnosis (BMI-adjusted P = 0.014 to 0.032). Also, one IGFBP1 and four IGFBP3 SNPs showed nominal genotypic association with T2D-ESRD (P = 0.002-0.049). After correcting for multiple tests, only rs730497 remanined significant., Conclusion: Variant rs730947 in the GCK1 gene appears to play a role in early ESRD onset in African Americans.

Background: The purpose of this study was to examine the effect of insulin on expression and synthesis of IGFBP-1 and IGFBP-2 in the baboon endometrium in vitro., Methods: Baboon endometrial explants collected from cycling, ovariectomized, steroid-treated, simulated-pregnant and pregnant animals were cultured for 48 h in the presence or absence of insulin, with or without estradiol, progesterone and hCG., Results: Insulin clearly inhibited IGFBP-1 production and mRNA expression in a time- and dose-dependent manner, whereas IGFBP-2 synthesis was not significantly affected. The inhibitory effects of insulin on IGFBP-1 were more evident in explants of non-pregnant tissue or tissue away from the implantation site. In the absence of insulin, synthesis of IGFBP-1 was induced in explants with low levels of de novo synthesis whereas IGFBP-2 synthesis was inhibited. This effect was potentiated by steroids and hCG in the explant cultures., Conclusion: Insulin differentially regulates endometrial IGFBP-1 and IGFBP-2 secretion in the baboon.

Background: Pancreatic carcinoma usually is a fatal disease with no cure, mainly due to its invasion and metastasis prior to diagnosis. We analyzed the gene expression profiles of paired primary pancreatic tumors and metastatic lesions from Ela-c-myc transgenic mice in order to identify genes that may be involved in the pancreatic cancer progression. Differentially expressed selected genes were verified by semi-quantitative and quantitative RT-PCR. To further evaluate the relevance of some of the selected differentially expressed genes, we investigated their expression pattern in human pancreatic cancer cell lines with high and low metastatic potentials., Results: Data indicate that genes involved in posttranscriptional regulation were a major functional category of upregulated genes in both primary pancreatic tumors (PT) and liver metastatic lesions (LM) compared to normal pancreas (NP). In particular, differential expression for splicing factors, RNA binding/pre-mRNA processing factors and spliceosome related genes were observed, indicating that RNA processing and editing related events may play critical roles in pancreatic tumor development and progression. High expression of insulin growth factor binding protein-1 (Igfbp1) and Serine proteinase inhibitor A1 (Serpina1), and low levels or absence of Wt1 gene expression were exclusive to liver metastatic lesion samples., Conclusion: We identified Igfbp1, Serpina1 and Wt1 genes that are likely to be clinically useful biomarkers for prognostic or therapeutic purposes in metastatic pancreatic cancer, particularly in pancreatic cancer where c-Myc is overexpressed.

Background: Hepatic expression of several gene products involved in glucose metabolism, including phosphoenolpyruvate carboxykinase (PEPCK), glucose-6-phosphatase (G6Pase) and insulin-like growth factor binding protein-1 (IGFBP-1), is rapidly and completely inhibited by insulin. This inhibition is mediated through the regulation of a DNA element present in each of these gene promoters, that we call the Thymine-rich Insulin Response Element (TIRE). The insulin signalling pathway that results in the inhibition of these gene promoters requires the activation of phosphatidylinositol 3-kinase (PI 3-kinase). However, the molecules that connect PI 3-kinase to these gene promoters are not yet fully defined. Glycogen Synthase Kinase 3 (GSK-3) is inhibited following activation of PI 3-kinase. We have shown previously that inhibitors of GSK-3 reduce the activity of two TIRE-containing gene promoters (PEPCK and G6Pase), whose products are required for gluconeogenesis., Results: In this report we demonstrate that in H4IIE-C3 cells, four distinct classes of GSK-3 inhibitor mimic the effect of insulin on a third TIRE-containing gene, IGFBP-1. We identify the TIRE as the minimum requirement for inhibition by these agents, and demonstrate that the target of GSK-3 is unlikely to be the postulated TIRE-binding protein FOXO-1. Importantly, overexpression of GSK-3 in cells reduces the insulin regulation of TIRE activity as well as endogenous IGFBP-1 expression., Conclusions: These results implicate GSK-3 as an intermediate in the pathway from the insulin receptor to the TIRE. Indeed, this is the first demonstration of an absolute requirement for GSK-3 inhibition in insulin regulation of gene transcription. These data support the potential use of GSK-3 inhibitors in the treatment of insulin resistant states such as Type 2 diabetes mellitus, but suggest that it will be important to identify all TIRE-containing genes to assess potential side effects of these agents.