A glucocorticoid- and diet-responsive pathway toggles adipocyte precursor cell activity in vivo. Science signaling 2016; 9 (451): ra103-?
Obesity is driven by excess caloric intake, which leads to the expansion of adipose tissue by hypertrophy and hyperplasia. Adipose tissue hyperplasia results from the differentiation of adipocyte precursor cells (APCs) that reside in adipose depots. Investigation into this process has elucidated a network of mostly transcription factors that drive APCs through the differentiation process. Using in vitro and in vivo approaches, our study revealed a signaling pathway that inhibited the initiation of the adipocyte differentiation program. Mouse adipocytes secreted the extracellular protease ADAMTS1, which triggered the production of the cytokine pleiotrophin (PTN) through the Wnt/-catenin pathway, and promoted proliferation rather than differentiation of APCs. Glucocorticoid exposure in vitro or in vivo reduced ADAMTS1 abundance in adipocytes. In addition, mice fed a high-fat diet showed decreased Adamts1 expression in the visceral perigonadal adipose depot, which expanded by adipogenesis in response to the diet, and increased Adamts1 expression in the subcutaneous inguinal adipose depot, which did not induce adipogenesis. Similar to what occurred in mouse subcutaneous adipose tissue, diet-induced weight gain increased the expression of ADAMTS1, PTN, and certain Wnt target genes in the subcutaneous adipose depot of human volunteers, suggesting the relevance of this pathway to physiological adipose tissue homeostasis and the pathogenesis of obesity. Thus, this pathway functions as a toggle on APCs, regulating a decision between differentiation and proliferation and coordinating the response of adipose tissue to systemic cues.
View details for PubMedID 27811141
Vitamin D Regulates Fatty Acid Composition in Subcutaneous Adipose Tissue Through Elovl3. Endocrinology 2016; 157 (1): 91-97
Fatty acids (FAs) are a major energy source in the body. White adipose tissue (WAT) is a primary site where FAs are stored as triacylglycerols. Brown adipose tissue (BAT) also stores and recruits FAs as a carbon source for uncoupled -oxidation during thermogenesis. The deletion of the vitamin D nuclear hormone receptor (VDR) gene in mice (VDRKO) results in a lean WAT phenotype with increased levels of expression of the BAT marker Ucp1 in the WAT. Yet, the impact of vitamin D/ VDR on FA composition in WAT has not been explored in detail. To address this question, we examined the FA composition of subcutaneous and visceral white adipose depots of VDRKO mice. We found that the levels of a subset of saturated and monounsaturated fatty acids of C18-C24 are specifically increased in the subcutaneous adipose depot in VDRKO mice. We revealed that a specific elongase enzyme (Elovl3), that has an important role in brown fat biology, is directly regulated by VDR and likely contributes to the altered FA composition in VDRKO mice. We also demonstrate that Elovl3 is regulated by vitamin D in vivo and tissue specifically in the subcutaneous WAT depot. We discovered that regulation of Elovl3 expression is mediated by ligand-dependent VDR occupancy of a negative response element in the promoter proximal region of the Elovl3 gene. These data suggest that vitamin D-VDR tissue specifically modulates FA composition in subcutaneous WAT through direct regulation of Elovl3 expression.
View details for DOI 10.1210/en.2015-1674
View details for PubMedID 26488808
Characterization of Cre Recombinase Activity for In Vivo Targeting of Adipocyte Precursor Cells STEM CELL REPORTS 2014; 3 (6): 1147-1158
The increased incidence of obesity and metabolic disease underscores the importance of elucidating the biology of adipose tissue development. The recent discovery of cell surface markers for prospective identification of adipose precursor cells (APCs) invivo will greatly facilitate these studies, yet tools for specifically targeting these cells invivo have not been identified. Here, we survey three transgenic mouse lines, Fabp4-Cre, PdgfR-Cre, and Prx1-Cre, precisely assessing Cre-mediated recombination in adipose stromal populations and mature tissues. Our data provide key insights into the utility of these tools to modulate gene expression in adipose tissues. In particular, Fabp4-Cre is not effective to target APCs, nor is its activity restricted to these cells. PdgfR-Cre directs recombination in the vast majority of APCs, but also targets other populations. In contrast, adipose expression of Prx1-Cre is chiefly limited to subcutaneous inguinal APCs, which will be valuable for dissection of APC functions among adipose depots.
View details for DOI 10.1016/j.stemcr.2014.10.009
View details for Web of Science ID 000346159300018
A plasmonic chip for biomarker discovery and diagnosis of type 1 diabetes NATURE MEDICINE 2014; 20 (8): 948-953
Type 1 diabetes (T1D) is an autoimmune disease, whereas type 2 diabetes (T2D) results from insulin resistance and beta cell dysfunction. Previously, the onset of these two separate diseases was easily distinguished, with children being most at risk for T1D and T2D occurring in overweight adults. However, the dramatic rise in obesity, coupled with the notable increase in T1D, has created a large overlap in these previously discrete patient populations. Delayed diagnosis of T1D can result in severe illness or death, and rapid diagnosis of T1D is critical for the efficacy of emerging therapies. However, attempts to apply next-generation platforms have been unsuccessful for detecting diabetes biomarkers. Here we describe the development of a plasmonic gold chip for near-infrared fluorescence-enhanced (NIR-FE) detection of islet cell-targeting autoantibodies. We demonstrate that this platform has high sensitivity and specificity for the diagnosis of T1D and can be used to discover previously unknown biomarkers of T1D.
View details for DOI 10.1038/nm.3619
View details for Web of Science ID 000340074600028
View details for PubMedID 25038825
The role of vitamin D in reducing cancer risk and progression NATURE REVIEWS CANCER 2014; 14 (5): 342-357
Vitamin D is not really a vitamin but the precursor to the potent steroid hormone calcitriol, which has widespread actions throughout the body. Calcitriol regulates numerous cellular pathways that could have a role in determining cancer risk and prognosis. Although epidemiological and early clinical trials are inconsistent, and randomized control trials in humans do not yet exist to conclusively support a beneficial role for vitamin D, accumulating results from preclinical and some clinical studies strongly suggest that vitamin D deficiency increases the risk of developing cancer and that avoiding deficiency and adding vitamin D supplements might be an economical and safe way to reduce cancer incidence and improve cancer prognosis and outcome.
View details for DOI 10.1038/nrc3691
View details for Web of Science ID 000335562000012
Circadian Rhythm Gene Period 3 Is an Inhibitor of the Adipocyte Cell Fate JOURNAL OF BIOLOGICAL CHEMISTRY 2011; 286 (11): 9063-9070
Glucocorticoids rapidly and robustly induce cell fate decisions in various multipotent cells, although the precise mechanisms of these important cellular events are not understood. Here we showed that glucocorticoids repressed Per3 expression and that this repression was critical for advancing mesenchymal stem cells to the adipocyte fate. Exogenous expression of Per3 inhibited adipogenesis, whereas knocking out Per3 enhanced that fate. Moreover, we found that PER3 formed a complex with PPAR? and inhibited PPAR?-mediated transcriptional activation via Ppar? response elements. Consistent with these findings, Per3 knock-out mice displayed alterations in body composition, with both increased adipose and decreased muscle tissue compared with wild-type mice. Our findings identify Per3 as potent mediator of cell fate that functions by altering the transcriptional activity of PPAR?.
View details for DOI 10.1074/jbc.M110.164558
View details for Web of Science ID 000288247700037
View details for PubMedID 21228270
Glucocorticoid regulation of the circadian clock modulates glucose homeostasis PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA 2009; 106 (41): 17582-17587
Circadian clock genes are regulated by glucocorticoids; however, whether this regulation is a direct or secondary effect and the physiological consequences of this regulation were unknown. Here, we identified glucocorticoid response elements (GREs) at multiple clock genes and showed that 3 were directly regulated by the glucocorticoid receptor. We determined that a GRE within the core clock gene Per2 was continuously occupied during rhythmic expression and essential for glucocorticoid regulation of that gene in vivo. We further demonstrated that mice with a genomic deletion spanning this GRE expressed elevated leptin levels and were protected from glucose intolerance and insulin resistance on glucocorticoid treatment but not from muscle wasting. We conclude that Per2 is an integral component of a particular glucocorticoid regulatory pathway and that glucocorticoid regulation of the peripheral clock is selectively required for some actions of glucocorticoids.
View details for DOI 10.1073/pnas.0909733106
View details for Web of Science ID 000270754400065
View details for PubMedID 19805059
Nephrogenic syndrome of inappropriate antidiuresis NEW ENGLAND JOURNAL OF MEDICINE 2005; 352 (18): 1884-1890
The syndrome of inappropriate antidiuretic hormone secretion (SIADH) is a common cause of hyponatremia. We describe two infants whose clinical and laboratory evaluations were consistent with the presence of SIADH, yet who had undetectable arginine vasopressin (AVP) levels. We hypothesized that they had gain-of-function mutations in the V2 vasopressin receptor (V2R). DNA sequencing of each patient's V2R gene (AVPR2) identified missense mutations in both, with resultant changes in codon 137 from arginine to cysteine or leucine. These novel mutations cause constitutive activation of the receptor and are the likely cause of the patients' SIADH-like clinical picture, which we have termed "nephrogenic syndrome of inappropriate antidiuresis."
View details for Web of Science ID 000228858600008
View details for PubMedID 15872203
Amphetamines promote mitochondrial dysfunction and DNA damage in pulmonary hypertension. JCI insight 2017; 2 (2): e90427
Amphetamine (AMPH) or methamphetamine (METH) abuse can cause oxidative damage and is a risk factor for diseases including pulmonary arterial hypertension (PAH). Pulmonary artery endothelial cells (PAECs) from AMPH-associated-PAH patients show DNA damage as judged by H2AX foci and DNA comet tails. We therefore hypothesized that AMPH induces DNA damage and vascular pathology by interfering with normal adaptation to an environmental perturbation causing oxidative stress. Consistent with this, we found that AMPH alone does not cause DNA damage in normoxic PAECs, but greatly amplifies DNA damage in hypoxic PAECs. The mechanism involves AMPH activation of protein phosphatase 2A, which potentiates inhibition of Akt. This increases sirtuin 1, causing deacetylation and degradation of HIF1, thereby impairing its transcriptional activity, resulting in a reduction in pyruvate dehydrogenase kinase 1 and impaired cytochrome c oxidase 4 isoform switch. Mitochondrial oxidative phosphorylation is inappropriately enhanced and, as a result of impaired electron transport and mitochondrial ROS increase, caspase-3 is activated and DNA damage is induced. In mice given binge doses of METH followed by hypoxia, HIF1 is suppressed and pulmonary artery DNA damage foci are associated with worse pulmonary vascular remodeling. Thus, chronic AMPH/METH can induce DNA damage associated with vascular disease by subverting the adaptive responses to oxidative stress.
View details for DOI 10.1172/jci.insight.90427
View details for PubMedID 28138562
Vitamin D mitigates the adverse effects of obesity on breast cancer in mice ENDOCRINE-RELATED CANCER 2016; 23 (4): 251-264
Obesity is an established risk factor for postmenopausal breast cancer (BCa), insulin resistance and vitamin D deficiency, and all contribute to increased synthesis of mammary estrogens, the drivers of estrogen receptor-positive (ER+) BCa growth. Since both dietary vitamin D and calcitriol treatments inhibit breast estrogen synthesis and signaling, we hypothesized that vitamin D would be especially beneficial in mitigating the adverse effects of obesity on ER+ BCa. To assess whether obesity exerted adverse effects on BCa growth and whether vitamin D compounds could reduce these unfavorable effects, we employed a diet-induced obesity (DIO) model in ovariectomized C57BL/6 mice. Breast tumor cells originally from syngeneic Mmtv-wnt1 transgenic mice were then implanted into the mammary fat pads of lean and obese mice. DIO accelerated the initiation and progression of the mammary tumors. Treatments with either calcitriol or dietary vitamin D reduced the adverse effects of obesity causing a delay in tumor appearance and inhibiting continued tumor growth. Beneficial actions of treatments with vitamin D or calcitriol on BCa and surrounding adipose tissue included: repressed Er, aromatase and Cox-2 expression, decreased tumor derived estrogen and PGE2 and reduced expression of leptin receptors and increased adiponectin receptors. We demonstrate that vitamin D treatments decreased insulin resistance, reduced leptin and increased adiponectin signaling and also regulated the LKB1/ AMPK pathway contributing to an overall decrease in local estrogen synthesis in the obese mice. We conclude that calcitriol and dietary vitamin D, acting by multiple interrelated pathways, mitigate obesity enhanced BCa growth in a postmenopausal setting.
View details for DOI 10.1530/ERC-15-0557
View details for Web of Science ID 000377691700007
View details for PubMedID 26817629
Tumor Autonomous Effects of Vitamin D Deficiency Promote Breast Cancer Metastasis. Endocrinology 2016; 157 (4): 1341-1347
Patients with breast cancer (BCa) frequently have preexisting vitamin D deficiency (low serum 25-hydroxyvitamin D) when their cancer develops. A number of epidemiological studies show an inverse association between BCa risk and vitamin D status in humans, although some studies have failed to find an association. In addition, several studies have reported that BCa patients with vitamin D deficiency have a more aggressive molecular phenotype and worse prognostic indicators. However, it is unknown whether this association is mechanistically causative and, if so, whether it results from systemic or tumor autonomous effects of vitamin D signaling. We found that ablation of vitamin D receptor expression within BCa cells accelerates primary tumor growth and enables the development of metastases, demonstrating a tumor autonomous effect of vitamin D signaling to suppress BCa metastases. We show that vitamin D signaling inhibits the expression of the tumor progression gene Id1, and this pathway is abrogated in vitamin D deficiency in vivo in 2 murine models of BCa. These findings are relevant to humans, because we discovered that the mechanism of VDR regulation of Inhibitor of differentiation 1 (ID1) is conserved in human BCa cells, and there is a negative correlation between serum 25-hydroxyvitamin D levels and the level of ID1 in primary tumors from patients with BCa.
View details for DOI 10.1210/en.2015-2036
View details for PubMedID 26934299
Vitamin D Mitigates the Adverse Effects of Obesity on Breast Cancer Growth in Mice Endocrine Related Cancer 2016
Inhibition of Mouse Breast Tumor-Initiating Cells by Calcitriol and Dietary Vitamin D MOLECULAR CANCER THERAPEUTICS 2015; 14 (8): 1951-1961
The anti-cancer actions of vitamin D and its hormonally active form, calcitriol, have been extensively documented in clinical and pre-clinical studies. However, the mechanisms underlying these actions have not been completely elucidated. Here we examined the effect of dietary vitamin D and calcitriol on mouse breast tumor-initiating cells (TICs, also known as cancer stem cells). We focused on MMTV-Wnt1 mammary tumors, for which markers for isolating TICs have previously been validated. We confirmed that these tumors expressed functional vitamin D receptors (VDRs) and estrogen receptors (ERs) and exhibited calcitriol-induced molecular responses including ER down-regulation. Following orthotopic implantation of MMTV-Wnt1 mammary tumor cells into mice, calcitriol injections or a vitamin D-supplemented diet caused a striking delay in tumor appearance and growth while a vitamin D-deficient diet accelerated tumor appearance and growth. Calcitriol inhibited TIC tumor spheroid formation in a dose-dependent manner in primary cultures and inhibited TIC self-renewal in secondary passages. A combination of calcitriol and ionizing radiation inhibited spheroid formation more than either treatment alone. Further, calcitriol significantly decreased TIC frequency as evaluated by in vivo limiting dilution analyses. Calcitriol inhibition of TIC spheroid formation could be overcome by the overexpression of -catenin, suggesting that the inhibition of Wnt/-catenin pathway is an important mechanism mediating the TIC inhibitory activity of calcitriol in this tumor model. Our findings indicate that vitamin D compounds target breast TICs reducing tumor-initiating activity. Our data also suggest that combining vitamin D compounds with standard therapies will enhance anti-cancer activity and may improve therapeutic outcomes.
View details for DOI 10.1158/1535-7163.MCT-15-0066
View details for Web of Science ID 000359324600018
Using SRM-MS to quantify nuclear protein abundance differences between adipose tissue depots of insulin-resistant mice JOURNAL OF LIPID RESEARCH 2015; 56 (5): 1068-1078
Insulin resistance underlies metabolic disease. Visceral, but not subcutaneous, white adipose tissue (WAT) has been linked to the development of insulin resistance, potentially due to differences in regulatory protein abundance. Here we investigate how protein levels are changed in insulin resistance in different WAT depots by developing a targeted proteomics approach to quantitatively compare the abundance of 42 nuclear proteins in subcutaneous and visceral WAT from a commonly-used insulin resistant mouse model, Lepr(db/db), and from C57BL/6J control mice. The most differentially-expressed proteins were important in adipogenesis, as confirmed by siRNA-mediated depletion experiments, suggesting a defect in adipogenesis in visceral, but not subcutaneous, insulin-resistant WAT. Furthermore, differentiation of visceral, but not subcutaneous, insulin-resistant stromal vascular cells (SVC) was impaired. In an in vitro approach to understand the cause of this impaired differentiation, we compared insulin-resistant visceral SVC to preadipocyte cell culture models made insulin-resistant by different stimuli. The insulin-resistant visceral SVC protein abundance profile correlated most with preadipocyte cell culture cells treated with both palmitate and TNF. Together, our study introduces a method to simultaneously measure and quantitatively compare nuclear protein expression patterns in primary adipose tissue and adipocyte cell cultures, which we show can reveal relationships between differentiation and disease states of different adipocyte tissue types.
View details for DOI 10.1194/jlr.D056317
View details for Web of Science ID 000353767200012
View details for PubMedID 25840986
BMPR2 Preserves Mitochondrial Function and DNA during Reoxygenation to Promote Endothelial Cell Survival and Reverse Pulmonary Hypertension CELL METABOLISM 2015; 21 (4): 596-608
Mitochondrial dysfunction, inflammation, and mutant bone morphogenetic protein receptor 2 (BMPR2) areassociated with pulmonary arterial hypertension (PAH), an incurable disease characterized by pulmonary arterial (PA) endothelial cell (EC) apoptosis, decreased microvessels, and occlusive vascular remodeling. We hypothesized that reduced BMPR2 induces PAEC mitochondrial dysfunction, promoting a pro-inflammatory or pro-apoptotic state. Mice with EC deletion of BMPR2 develop hypoxia-induced pulmonary hypertension that, in contrast to non-transgenic littermates, does not reverse upon reoxygenation and is associated with reduced PA microvessels and lung EC p53, PGC1 and TFAM, regulators of mitochondrial biogenesis, and mitochondrial DNA. Decreasing PAEC BMPR2 by siRNA during reoxygenation represses p53, PGC1, NRF2, TFAM, mitochondrial membrane potential, and ATP and induces mitochondrial DNA deletion and apoptosis. Reducing PAEC BMPR2 in normoxia increases p53, PGC1, TFAM, mitochondrial membrane potential, ATP production, and glycolysis, and induces mitochondrial fission and a pro-inflammatory state. These features are recapitulated in PAECs from PAH patients with mutant BMPR2.
View details for DOI 10.1016/j.cmet.2015.03.010
View details for Web of Science ID 000352500800014
The development of next-generation screening and diagnostic platforms will change diabetes care. Expert review of molecular diagnostics 2015; 15 (3): 291-294
Diabetes mellitus is a common disease with a rising incidence and the findings of hyperglycemia and glucosuria. However, there are multiple types of diabetes, each with distinct etiologies. The two major types of diabetes are type 1, which is caused by an autoimmune process, and type 2, which is thought to be primarily metabolic, resulting from insulin resistance, often in the setting of obesity. Historically, the distinction between these two types was obvious. Here, we discuss how this paradigm has dramatically changed because of both the evolving epidemiology of diabetes mellitus and new and emerging tools, and therapies to diagnose and treat diabetes. As we believe that understanding these changes is critical to providing optimal care to patients with diabetes, we have developed a novel plasmonic gold chip platform that is able to meet the new and emerging demands of modern diabetes care.
View details for DOI 10.1586/14737159.2015.1002468
View details for PubMedID 25583407
Novel GATA6 Mutations in Patients with Pancreatic Agenesis and Congenital Heart Malformations PLOS ONE 2015; 10 (2)
Mifepristone Treatment of Cushings Syndrome in a Pediatric Patient Pediatrics 2015; 136 (5)
Cell-Autonomous Regulation of Brown Fat Identity Gene UCP1 by Unliganded Vitamin D Receptor. Molecular endocrinology 2013; 27 (10): 1632-1642
White adipose tissue stores energy in the form of lipids, and brown adipose tissue expends energy via uncoupled fatty acid oxidation, which leads to the generation of heat. Obesity reflects an imbalance between energy storage and energy expenditure and is strongly associated with metabolic and cardiovascular disease. Therefore, there are important medical and biological implications for elucidating the mechanisms that promote energy expenditure in humans. Animal models with altered vitamin D receptor (VDR) expression have changes in energy expenditure. However, the specific mechanism for this effect has not been elucidated and the relevance for humans is unclear. Here we show, using human patient samples from individuals with hereditary vitamin D resistant rickets, that the VDR directly inhibits the expression of uncoupling protein-1 (UCP1), the critical protein for uncoupling fatty acid oxidation in brown fat and burning energy. The inhibition is enforced by VDR occupancy of a negative response element in the promoter proximal region of the UCP1 gene. Deletion of VDR increases UCP1 expression and results in a "browning" of adipocytes. Importantly, we found that this process occurs cell autonomously and is independent of the physiologic VDR hormone ligand, 1,25-dihydroxyvitamin D. These results identify a mechanism for modulating energy balance in humans.
View details for DOI 10.1210/me.2013-1037
View details for PubMedID 23906633
Characterization of Three Vasopressin Receptor 2 Variants: An Apparent Polymorphism (V266A) and Two Loss-of-Function Mutations (R181C and M311V) PLOS ONE 2013; 8 (6)
Arginine vasopressin (AVP) is released from the posterior pituitary and controls water homeostasis. AVP binding to vasopressin V2 receptors (V2Rs) located on kidney collecting duct epithelial cells triggers activation of Gs proteins, leading to increased cAMP levels, trafficking of aquaporin-2 water channels, and consequent increased water permeability and antidiuresis. Typically, loss-of-function V2R mutations cause nephrogenic diabetes insipidus (NDI), whereas gain-of-function mutations cause nephrogenic syndrome of inappropriate antidiuresis (NSIAD). Here we provide further characterization of two mutant V2Rs, R181C and M311V, reported to cause complete and partial NDI respectively, together with a V266A variant, in a patient diagnosed with NSIAD. Our data in HEK293FT cells revealed that for cAMP accumulation, AVP was about 500- or 30-fold less potent at the R181C and M311V mutants than at the wild-type receptor respectively (and about 4000- and 60-fold in COS7 cells respectively). However, in contrast to wild type V2R, the R181C mutant failed to increase inositol phosphate production, while with the M311V mutant, AVP exhibited only partial agonism in addition to a 37-fold potency decrease. Similar responses were detected in a BRET assay for -arrestin recruitment, with the R181C receptor unresponsive to AVP, and partial agonism with a 23-fold decrease in potency observed with M311V in both HEK293FT and COS7 cells. Notably, the V266A V2R appeared functionally identical to the wild-type receptor in all assays tested, including cAMP and inositol phosphate accumulation, -arrestin interaction, and in a BRET assay of receptor ubiquitination. Each receptor was expressed at comparable levels. Hence, the M311V V2R retains greater activity than the R181C mutant, consistent with the milder phenotype of NDI associated with this mutant. Notably, the R181C mutant appears to be a Gs protein-biased receptor incapable of signaling to inositol phosphate or recruiting -arrestin. The etiology of NSIAD in the patient with V266A V2R remains unknown.
View details for DOI 10.1371/journal.pone.0065885
View details for Web of Science ID 000321099000116
View details for PubMedID 23762448
Adipose circadian clocks: coordination of metabolic rhythms by clock genes, steroid hormones, and PPARs. Hormone molecular biology and clinical investigation 2013; 14 (1): 15-24
Abstract A central clock consisting of interconnected positive and negative feedback gene loops operates in the brain, tying rhythmic activity to the 24-h day. The central clock entrains similar feedback loops present in most peripheral tissues to coordinate metabolic gene expression among organs and with feeding activity for more efficient utilization of resources. Recent studies are beginning to elucidate the intricate feedback mechanisms among central and peripheral clocks and their roles in activity and metabolic homeostasis. Adipose tissue serves as a major energy storage organ and releases paracrine and endocrine hormones to signal energy status to other organs. Within the adipose tissue, the transcriptional feedback regulation between clock genes and nuclear hormone receptors, together with direct protein associations among these molecules, ensures the expression of metabolic genes at the appropriate time. This review will summarize the important components and mechanisms of adipose clock entrainment, particularly highlighting instructive studies carried out in mice. This research not only illustrates the intricate connections between clocks and metabolism but also provides potential mechanisms to correct abnormalities induced by disrupted sleep or poor diet.
View details for DOI 10.1515/hmbci-2013-0011
View details for PubMedID 25436716
Enhanced BRET Technology for the Monitoring of Agonist-Induced and Agonist-Independent Interactions between GPCRs and -Arrestins. Frontiers in endocrinology 2010; 1: 12-?
The bioluminescence resonance energy transfer (BRET) technique has become extremely valuable for the real-time monitoring of protein-protein interactions in live cells. This method is highly amenable to the detection of G protein-coupled receptor (GPCR) interactions with proteins critical for regulating their function, such as ?-arrestins. Of particular interest to endocrinologists is the ability to monitor interactions involving endocrine receptors, such as orexin receptor 2 or vasopressin type II receptor. The BRET method utilizes heterologous co-expression of fusion proteins linking one protein of interest (GPCR) to a bioluminescent donor enzyme, a variant of Renilla luciferase, and a second protein of interest (?-arrestin) to an acceptor fluorophore. If in close proximity, energy resulting from oxidation of the coelenterazine substrate by the donor will transfer to the acceptor, which in turn fluoresces. Using novel luciferase constructs, we were able to monitor interactions not detectable using less sensitive BRET combinations in the same configuration. In particular, we were able to show receptor/?-arrestin interactions in an agonist-independent manner using Rluc8-tagged mutant receptors, in contrast to when using Rluc. Therefore, the enhanced BRET methodology has not only enabled live cell compound screening as we have recently published, it now provides a new level of sensitivity for monitoring specific transient, weak or hardly detectable protein-protein complexes, including agonist-independent GPCR/?-arrestin interactions. This has important implications for the use of BRET technologies in endocrine drug discovery programs as well as academic research.
View details for DOI 10.3389/fendo.2010.00012
View details for PubMedID 22654789
Agonist-Independent Interactions between beta-Arrestins and Mutant Vasopressin Type II Receptors Associated with Nephrogenic Syndrome of Inappropriate Antidiuresis MOLECULAR ENDOCRINOLOGY 2009; 23 (4): 559-571
Nephrogenic syndrome of inappropriate antidiuresis is a recently identified genetic disease first described in two unrelated male infants with severe symptomatic hyponatremia. Despite undetectable arginine vasopressin levels, patients have inappropriately concentrated urine resulting in hyponatremia, hypoosmolality, and natriuresis. It was found that each infant had a different mutation of the vasopressin type II receptor (V2R) at codon 137 where arginine was converted to cysteine or leucine (R137C or R137L), resulting in constitutive signaling. Interestingly, a missense mutation at the same codon, converting arginine to histidine (R137H), leads to the opposite disease phenotype with a loss of the kidney's ability to concentrate urine resulting in nephrogenic diabetes insipidus. This mutation is associated with impaired signaling, although whether this is predominantly due to impaired trafficking to the plasma membrane, agonist-independent internalization, or G protein uncoupling is currently unclear. Using bioluminescence resonance energy transfer and confocal microscopy, we demonstrate that both V2R-R137C and V2R-R137L mutants interact with beta-arrestins in an agonist-independent manner resulting in dynamin-dependent internalization. This phenotype is similar to that observed for V2R-R137H, which is intriguing considering that it is accompanied by constitutive rather than impaired signaling. Consequently, it would seem that agonist-independent internalization per se is unlikely to be the major determinant of impaired V2R-R137H signaling. Our findings indicate that the V2R-R137C and V2R-R137L mutants traffic considerably more efficiently to the plasma membrane than V2R-R137H, identifying this as a potentially important mutation-dependent difference affecting V2R function.
View details for DOI 10.1210/me.2008-0321
View details for Web of Science ID 000264707000014
View details for PubMedID 19179480
Glucocorticoids Influence on Mesenchymal Stem Cells and Implications for Metabolic Disease PEDIATRIC RESEARCH 2009; 65 (2): 249-251
Metabolic disease is a well established major public health problem in the adult population. However, the origins of metabolic disease of adults can begin early in life. In addition, in recent years, there has been a disturbing increase in the number of children developing the full presentation of metabolic disease as a result of the increase in obesity in this population. Therefore, pediatricians and pediatric physician-scientists are essential both for instituting preventive measures and developing new therapies. This challenge has been met with a substantial increase in research into both the clinical and basic science of metabolism. A connection between glucocorticoids and the origins of metabolic disease is one enticing clue because of the clinical similarity between patients with glucocorticoid excess and those with metabolic disease. This perspective highlights one series of investigations that has advanced our understanding of the development of metabolic disease. In this work, a unifying link was found by investigating the role of glucocorticoids on cell fate and differentiation of mesenchymal stem cells. We conclude that elucidating the mechanisms by which glucocorticoids modulate cell fate decisions holds promise for developing new therapies and preventative measures.
View details for Web of Science ID 000262766100023
View details for PubMedID 19262295
Conservation analysis predicts in vivo occupancy of glucocorticoid receptor-binding sequences at glucocorticoid-induced genes PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA 2008; 105 (15): 5745-5749
The glucocorticoid receptor (GR) interacts with specific GR-binding sequences (GBSs) at glucocorticoid response elements (GREs) to orchestrate transcriptional networks. Although the sequences of the GBSs are highly variable among different GREs, the precise sequence within an individual GRE is highly conserved. In this study, we examined whether sequence conservation of sites resembling GBSs is sufficient to predict GR occupancy of GREs at genes responsive to glucocorticoids. Indeed, we found that the level of conservation of these sites at genes up-regulated by glucocorticoids in mouse C3H10T1/2 mesenchymal stem-like cells correlated directly with the extent of occupancy by GR. In striking contrast, we failed to observe GR occupancy of GBSs at genes repressed by glucocorticoids, despite the occurrence of these sites at a frequency similar to that of the induced genes. Thus, GR occupancy of the GBS motif correlates with induction but not repression, and GBS conservation alone is sufficient to predict GR occupancy and GRE function at induced genes.
View details for DOI 10.1073/pnas.0801551105
View details for Web of Science ID 000255237200019
View details for PubMedID 18408151
Is your metabolism determined by (cell) fate? PEDIATRIC RESEARCH 2007; 61 (6): 636-639
The rise in the incidence of metabolic disease to become a major public health problem has been met with a substantial increase in research into both the clinical and basic science of metabolism. This work has revealed that the origins of metabolic diseases of adults can begin early in life. Furthermore, the age of onset of symptoms has been rapidly decreasing. Therefore, pediatricians should be critically involved in both the generation of new therapies as well as the institution of measures of disease prevention. This perspective examines how recent advances have improved our understanding of the development of metabolic diseases. A connection between glucocorticoids and the origins of metabolic disease is one enticing clue because of the clinical similarity between patients with glucocorticoid excess and those with metabolic disease. A unifying link was found by investigating the role of glucocorticoids on cell fate and differentiation of mesenchymal stem cells. We conclude that understanding the mechanisms by which glucocorticoids can modify how cell fate decisions are made holds promise for developing new therapies and preventative measures.
View details for DOI 10.1203/pdr.0b013e31805d854e
View details for Web of Science ID 000246787300003
View details for PubMedID 17426662
Cardiac troponin increases among runners in the Boston Marathon ANNALS OF EMERGENCY MEDICINE 2007; 49 (2): 137-143
Studies indicate that running a marathon can be associated with increases in serum cardiac troponin levels. The clinical significance of such increases remains unclear. We seek to determine the prevalence of troponin increases and epidemiologic factors associated with these increases in a large and heterogeneous cohort of marathon finishers.Entrants in the 2002 Boston Marathon were recruited 1 to 2 days before the race. Data collected included demographic and training history, symptoms experienced during the run, and postrace troponin T and I levels. Simple descriptive statistics were performed to describe the prevalence of troponin increases and runner characteristics.Of 766 runners enrolled, 482 had blood analyzed at the finish line. In all, 34% were women, 20% were younger than 30 years, and 92% had run at least 1 previous marathon. Most runners (68%) had some degree of postrace troponin increase (troponin T > or = 0.01 ng/mL or troponin I > or = 0.1 ng/mL), and 55 (11%) had significant increases (troponin T > or = 0.075 ng/mL or troponin I > or = 0.5 ng/mL). Running inexperience (< 5 previous marathons) and young age (< 30 years) were associated with elevated troponins. These correlates were robust throughout a wide range of troponin thresholds considered. Health factors, family history, training, race performance, and symptoms were not associated with increases.Troponin increases were relatively common among marathon finishers and can reach levels typically diagnostic for acute myocardial infarction. Less marathon experience and younger age appeared to be associated with troponin increases, whereas race duration and the presence of traditional cardiovascular risk factors were not. Further work is needed to determine the clinical significance of these findings.
View details for DOI 10.1016/j.annemergmed.2006.09.024
View details for Web of Science ID 000243957800002
View details for PubMedID 17145114
Report of a hurthle cell neoplasm in a peripubertal girl THYROID 2007; 17 (2): 175-178
Thyroid nodules are rare in children compared to adults. Although most thyroid nodules are benign, the risk of malignancy is greater in pediatric patients. Papillary and follicular cell tumors account for the majority of thyroid neoplasms; Hrthle cell tumors account for less than 5%. Despite being uncommon, malignant Hrthle cell tumors are potentially more aggressive than papillary and follicular cell tumors. Therefore, distinguishing between types of thyroid neoplasms in a timely fashion has implications for prognosis and therapy. We describe a 12-year-old peripubertal girl who presented with a large right-sided thyroid nodule that was subsequently diagnosed as a Hrthle cell adenoma. To our knowledge, she represents the youngest patient with a Hrthle cell neoplasm.
View details for DOI 10.1089/thy.2006.0214
View details for Web of Science ID 000244642400014
View details for PubMedID 17316121
Gain-of-function mutations in the V2 vasopressin receptor HORMONE RESEARCH 2007; 67: 121-125
Nephrogenic syndrome of inappropriate antidiuresis (NSIAD): a paradigm for activating mutations causing endocrine dysfunction. Pediatric endocrinology reviews : PER 2006; 4: 66-70
Mutations of G protein-coupled receptors are responsible for a wide range of diseases. With respect to water balance and vasopressin signaling, more than 180 different inactivating mutations have been previously described in the V2 vasopressin receptor (V2R), resulting in nephrogenic diabetes insipidus. In contrast, we have recently described the first known patients with V2R activating mutations. Patients with these novel gain-of-function V2R mutations have a disorder which we have termed "nephrogenic syndrome of inappropriate antidiuresis" (NSIAD): a clinical presentation consistent with the syndrome of inappropriate antidiuretic hormone secretion but with undetectable levels of arginine vasopressin (antidiuretic hormone). The mechanisms by which these mutations constitutively activate the V2R are currently being investigated.
View details for PubMedID 17261972
Myostatin modulates adipogenesis to generate adipocytes with favorable metabolic effects PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA 2006; 103 (42): 15675-15680
A pluripotent cell line, C3H10T1/2, is induced to undergo adipogenesis by a mixture of factors that includes a glucocorticoid such as dexamethasone. We found that expression of myostatin (MSTN), a TGF-beta family member extensively studied in muscle, was induced by dexamethasone under those differentiation conditions. Moreover, MSTN could substitute for dexamethasone in the adipogenesis mixture. However, the adipocytes induced by MSTN in both cell culture and transgenic mice were small and expressed markers characteristic of immature adipocytes. These adipocytes exhibited cell-autonomous increases in insulin sensitivity and glucose oxidation. In mice, these effects produced elevated systemic insulin sensitivity and resistance to diet-induced obesity. Modulation of the final stages of adipogenesis may provide a novel approach to understanding and treating metabolic disease.
View details for DOI 10.1073/pnas.0607501103
View details for Web of Science ID 000241476200072
View details for PubMedID 17030820
Nephrogenic syndrome of inappropriate antidiuresis: A novel disorder in water balance in pediatric patients AMERICAN JOURNAL OF MEDICINE 2006; 119: S54-S58
The syndrome of inappropriate antidiuretic hormone secretion (SIADH) is a common cause of hyponatremia. We report findings in 2 unrelated male infants whose clinical presentation and laboratory findings were consistent with SIADH, but who exhibited unmeasurable arginine vasopressin (AVP) levels on repeated occasions. We hypothesized that these infants had a novel gain of function defect in the AVP-signaling pathway. DNA sequencing of each patient's vasopressin V2 receptor (V2R) gene identified mutations (R137C or R137L) in each. R137H mutations have been reported previously to cause nephrogenic diabetes insipidus. To further characterize the effects of these mutations, we re-created each mutation by site-directed mutagenesis in a vasopressin V2R expression vector and cotransfected COS-7 cells with wild-type and mutant vasopressin V2R vectors and a cyclic adenosine monophosphate-responsive luciferase reporter plasmid. The luciferase activity was induced 7.5-fold (R137L mutant; P = 0.0037) and 4-fold (R137C mutant; P = 0.013) more than the wild-type vasopressin V2R, which is the empty vector or the inactivating R137H mutant. This novel gain of function mutation in the vasopressin V2R can cause constitutive activation of the receptor and resultant hyponatremia. These findings represent a previously unrecognized genetic disease, which was designated as nephrogenic syndrome of inappropriate antidiuresis. A number of questions have emerged, including the following: (1) What is the frequency? (2) Are there nonrenal manifestations? (3) Are heterozygotes affected? (4) What is the optimal therapy? and (5) How do these mutations cause constitutive activation of the receptor?
View details for DOI 10.1016/j.amjmed.2006.05.008
View details for Web of Science ID 000239014700008
View details for PubMedID 16843086
Oral urea for the treatment of chronic syndrome of inappropriate antidiuresis in children JOURNAL OF PEDIATRICS 2006; 148 (1): 128-131
We report the successful use of oral urea in the management of children with chronic syndrome of inappropriate antidiuretic hormone secretion (SIAD). We performed a retrospective review of four children with chronic SIAD. After initial attempts at management with fluid restriction, each was started on a 30% to 50% oral urea solution, and the dose was titrated until normal serum sodium was achieved. Fluid intake was liberalized after serum sodium normalization. All four children normalized their serum sodium. No side effects or toxicities were experienced. Oral urea is a safe, effective treatment for chronic SIAD in children.
View details for DOI 10.1016/j.jpeds.2005.08.031
View details for Web of Science ID 000234954900028
View details for PubMedID 16423613
Nephrogenic syndrome of inappropriate antidiuresis - Reply NEW ENGLAND JOURNAL OF MEDICINE 2005; 353 (5): 530-530
Carbohydrate-deficient glycoprotein syndrome-associated pericardial effusion treated with corticosteroids and salicylic acid PEDIATRIC CARDIOLOGY 2002; 23 (4): 469-471
We describe an infant with a persistent pericardial effusion who was diagnosed with carbohydrate-deficient glycoprotein syndrome (CDGS)-Ia. She was born with mild dysmorphic features and common cardiac abnormalities. However, she re-presented at 2.5 months of age with a pericardial effusion. We decided to embark on a therapeutic trial of corticosteroids and salicylic acid therapy in an attempt to avoid pericardectomy. After 3 weeks of medical treatment the effusion resolved. This experience allows us to propose that medical management with corticosteroids and salicylic acid can be considered as an alternative to surgical therapy for CDGS-I patients with persistent pericardial effusions.
View details for DOI 10.1007/s00246-002-1497-1
View details for Web of Science ID 000176665700018
View details for PubMedID 12170369
The development of androgen-independent prostate cancer NATURE REVIEWS CANCER 2001; 1 (1): 34-45
The normal prostate and early-stage prostate cancers depend on androgens for growth and survival, and androgen ablation therapy causes them to regress. Cancers that are not cured by surgery eventually become androgen independent, rendering anti-androgen therapy ineffective. But how does androgen independence arise? We predict that understanding the pathways that lead to the development of androgen-independent prostate cancer will pave the way to effective therapies for these, at present, untreatable cancers.
View details for Web of Science ID 000180397000012
View details for PubMedID 11900250
A carboxy-terminal deletion mutant of Notch1 accelerates lymphoid oncogenesis in E2A-PBX1 transgenic mice BLOOD 2000; 96 (5): 1906-1913
PBX1 is a proto-oncogene that plays important roles in pattern formation during development. It was discovered as a fusion with the E2A gene after chromosomal translocations in a subset of acute leukemias. The resulting E2a-Pbx1 chimeric proteins display potent oncogenic properties that appear to require dimerization with Hox DNA binding partners. To define molecular pathways that may be impacted by E2a-Pbx1, a genetic screen consisting of neonatal retroviral infection was used to identify genes that accelerate development of T-cell tumors in E2A-PBX1 transgenic mice. Retroviral insertions in the Notch1 gene were observed in 88% of tumors arising with a shortened latency. Among these, approximately half created a Notch(IC) allele, encoding the intracellular, signaling portion of Notch1, suggesting a synergistic interaction between the Notch and E2a-Pbx1 pathways in oncogenesis. The remaining proviral insertions involving Notch1 occurred in a more 3' exon, resulting in truncating mutations that deleted the carboxy-terminal region of Notch1 containing negative regulatory sequences (Notch1(DeltaC)). In contrast to Notch(IC), forced expression of Notch1(DeltaC) in transgenic mice did not perturb thymocyte growth or differentiation. However, mice transgenic for both the E2A-PBX1 and Notch1(DeltaC) genes displayed a substantially shortened latency for tumor development compared with E2A-PBX1 single transgenic mice. These studies reveal a novel mechanism for oncogenic activation of Notch1 and demonstrate a collaborative relationship between 2 cellular oncogenes that also contribute to cell fate determination during embryonic development. (Blood. 2000;96:1906-1913)
View details for Web of Science ID 000089578300042
View details for PubMedID 10961893
Pim1 cooperates with E2A-PBX to facilitate the progression of the thymic lymphomas in transgenic mice. LIPPINCOTT WILLIAMS & WILKINS. 1998: 103A-103A
Pim1 cooperates with E2a-Pbx1 to facilitate the progression of thymic lymphomas in transgenic mice ONCOGENE 1997; 15 (22): 2735-2742
Mice transgenic for the leukemia oncogene E2A-PBX1 invariably develop lethal, high-grade T-cell lymphomas by 5 months of age. In this study, retroviral insertional mutagenesis was employed to identify oncogenes that cooperate with the E2A-PBX1 transgene in lymphomagenesis. Neonatal retroviral infection substantially reduced length of survival due to accelerated development of lymphomas (81 versus 130 days). The Pim1 gene was targeted by retroviral insertions in 48% of accelerated lymphomas whereas less than 5% contained activated c-Myc and none contained activated Pim2. However, Pim1 DNA rearrangements were frequently sub-stoichiometric and not present at all sites of involvement in an otherwise monoclonal lymphoma indicating that Pim1 activation occurred late in the course of lymphomagenesis. Tumor subpopulations containing activated Pim1 alleles displayed a substantial growth advantage over Pim1 negative cells following serial transfer to secondary, syngeneic recipients. Cooperative interactions were observed in intercrossed Pim1 and E2A-PBX1 transgenic mice in which all double transgenic progeny developed lethal, diffuse T lineage lymphomas by 3 months of age, whereas only 13% of E2A-PBX1 and none of Pim1 single transgenic intercross progeny developed lymphomas by 1 year. Tumors from double transgenic mice were monoclonal providing evidence that additional genetic events were required for transformation. Therefore, Pim1 and E2a-Pbx1 cooperate in T lineage lymphomagenesis but they are not sufficient and the role of Pim1 is more likely to be associated with tumor progression.
View details for Web of Science ID A1997YH46800011
View details for PubMedID 9401000
The chimeric oncoproteins E2A-PBX1 and E2A-HLF are concentrated within spherical nuclear domains ONCOGENE 1997; 15 (17): 2059-2067
Oncogenic mutation of nuclear transcription factors often is associated with altered patterns of subcellular localization that may be of functional importance. The leukemogenic transcription factor gene E2A-PBX1 is created through fusion of the genes E2A and PBX1 as a result of t(1;19) in acute lymphoblastic leukemia. We evaluated subcellular localization patterns of E2A-PBX1 protein in transfected cells using immunofluorescence. Full-length E2A-PBX1 was exclusively nuclear and was concentrated in spherical domains denoted chimeric-E2A oncoprotein domains (CODs). In contrast, nuclear fluorescence for wild-type E2A or PBX1 proteins was diffuse. Enhanced concentrations of RNA polymerase II within many CODs and the requirement for an E2A-encoded activation domain suggested transcriptional relevance. However, in situ co-detection of nascent transcripts labeled with bromouridine failed to confirm altered transcriptional activity in relation to CODs. CODs also failed to co-localize with other proteins known to occupy functional nuclear compartments, including the transcription factor PML, the spliceosome-associated protein SC-35 and the adenovirus replication factor DBP, or with foci of DNA replication. Co-transfection of Hoxb7, a homeodomain protein capable of enhancing DNA binding by PBX1, impaired COD formation, suggesting that CODs contain E2A-PBX1 protein not associated with DNA. We conclude that, as a 'gain of function' phenomenon requiring protein elements from both E2A and PBX1, COD formation may be relevant to the biology of E2A-PBX1 in leukemogenesis.
View details for Web of Science ID A1997YC28400007
View details for PubMedID 9366523
A NOVEL ROLE FOR DNA PHOTOLYASE - BINDING TO DNA DAMAGED BY DRUGS IS ASSOCIATED WITH ENHANCED CYTOTOXICITY IN SACCHAROMYCES-CEREVISIAE MOLECULAR AND CELLULAR BIOLOGY 1994; 14 (12): 8071-8077
DNA photolyase binds to and repairs cyclobutane pyrimidine dimers induced by UV radiation. Here we demonstrate that in the yeast Saccharomyces cerevisiae, photolyase also binds to DNA damaged by the anticancer drugs cis-diamminedichloroplatinum (cis-DDP) and nitrogen mustard (HN2) and by the alkylating agent N-methyl-N'-nitro-N-nitrosoguanidine (MNNG). Surprisingly, mutations in photolyase were associated with resistance of yeast cells to cis-DDP, MNNG, 4-nitroquinoline oxide (4NQO), and HN2. Transformation of yeast photolyase mutants with the photolyase gene increased sensitivity to these agents. Thus, while the binding of photolyase to DNA damaged by UV radiation aids survival of the cell, binding to DNA damaged by other agents may interfere with cell survival, perhaps by making the lesions inaccessible to the nucleotide excision repair system.
View details for Web of Science ID A1994PV67400036
View details for PubMedID 7969145