Archive for the ‘Genomic Endocrinology’ Category

Obesity Variant Circuitry

Larry H. Bernstein, MD, FCAP, Curator



FTO Obesity Variant Circuitry and Adipocyte Browning in Humans

Melina Claussnitzer,  Simon N. Dankel, Kyoung-Han Kim,  Gerald Quon,  Wouter Meuleman,  Christine Haugen,  Viktoria Glunk,  Isabel S. Sousa, et al.

N Engl J Med 2015; 373:895-907  Sept 3, 2015    DOI: http://dx.10.org1/056/NEJMoa1502214


Genomewide association studies can be used to identify disease-relevant genomic regions, but interpretation of the data is challenging. The FTO region harbors the strongest genetic association with obesity, yet the mechanistic basis of this association remains elusive.

Full Text of Background…



We examined epigenomic data, allelic activity, motif conservation, regulator expression, and gene coexpression patterns, with the aim of dissecting the regulatory circuitry and mechanistic basis of the association between the FTO region and obesity. We validated our predictions with the use of directed perturbations in samples from patients and from mice and with endogenous CRISPR–Cas9 genome editing in samples from patients.

Full Text of Methods…



Our data indicate that the FTO allele associated with obesity represses mitochondrial thermogenesis in adipocyte precursor cells in a tissue-autonomous manner. The rs1421085 T-to-C single-nucleotide variant disrupts a conserved motif for the ARID5B repressor, which leads to derepression of a potent preadipocyte enhancer and a doubling of IRX3 and IRX5 expression during early adipocyte differentiation. This results in a cell-autonomous developmental shift from energy-dissipating beige (brite) adipocytes to energy-storing white adipocytes, with a reduction in mitochondrial thermogenesis by a factor of 5, as well as an increase in lipid storage. Inhibition of Irx3 in adipose tissue in mice reduced body weight and increased energy dissipation without a change in physical activity or appetite. Knockdown of IRX3 or IRX5 in primary adipocytes from participants with the risk allele restored thermogenesis, increasing it by a factor of 7, and overexpression of these genes had the opposite effect in adipocytes from nonrisk-allele carriers. Repair of the ARID5B motif by CRISPR–Cas9 editing of rs1421085 in primary adipocytes from a patient with the risk allele restored IRX3 and IRX5 repression, activated browning expression programs, and restored thermogenesis, increasing it by a factor of 7.


Effect of the FTO Locus on IRX3 and IRX5 in Human Adipocyte Progenitor Cells

To identify the cell types in which the causal variant may act, we examined chromatin state maps15,16 of the FTO obesity region across 127 cell types. An unusually long enhancer (12.8 kb) in mesenchymal adipocyte progenitors indicated a major regulatory locus (Figure 1B; and Fig. S1A, S1B, and S1C in the Supplementary Appendix). Haplotype-specific enhancer assays showed activity in association with the risk haplotype that was 2.4 times as high as that associated with the nonrisk haplotype in human SGBS adipocytes (i.e., adipocytes derived from a patient with the Simpson–Golabi–Behmel syndrome), which indicated genetic control of enhancer activity (Figure 1C). Enhancers in brain cells and other cell types were considerably shorter than those in mesenchymal adipocyte progenitors and lacked allelic activity (Fig. S1C and S1D in the Supplementary Appendix).


Figure 1. Activation of a Superenhancer in Human Adipocyte Progenitors by the FTO Obesity Risk Haplotype.

Panel A shows the genetic association with body-mass index (BMI) for all common FTO locus variants,14 including the reported single-nucleotide variant (SNV) rs1558902 (red diamond) and the predicted causal SNV rs1421085 (red square). Gray shading delineates consecutive 10-kb segments. CEU denotes a population of Utah residents with northern and western European ancestry, and LD linkage disequilibrium. Panel B shows chromatin state annotations for the locus across 127 reference epigenomes (rows) for cell and tissue types profiled by the Roadmap Epigenomics Project.15,16 For information on the colors used to denote chromatin states, see Figure S1A in the Supplementary Appendix. Vertical lines delineate the consecutive 10-kb segments shown in Panel A. ESC denotes embryonic stem cell, HSC hematopoietic stem cell, and iPSC induced pluripotent stem cell. Panel C shows human SGBS adipocyte enhancer activity, for 10-kb tiles, of the risk and nonrisk haplotypes with the use of relative luciferase expression. The boxes indicate means from seven triplicate experiments, and T bars indicate standard deviations.

To predict putative target genes, we examined large domains that had long-range three-dimensional chromatin interactions surrounding FTO and identified eight candidate genes (Figure 2A and 2B)



Figure 2. Activation of IRX3 and IRX5 Expression in Human Adipocyte Progenitors by the FTO Obesity Risk Genotype.

Panel A shows gene annotations and LD with array tag variant rs9930506 in a 2.5-Mb window; LD is expressed as r2 values in the CEU population. Arrows indicate the direction of transcription of annotated genes in the locus. Panel B shows chromosome conformation capture (Hi-C) interactions contact probabilities in human IMR90 myofibroblasts,22 revealing a 2-Mb topologically associating domain, and LD mean r2 statistics for all SNV pairs at 40-kb resolution. Panel C shows box plots for expression levels, after 2 days of differentiation, in human adipose progenitors isolated from 20 risk-allele carriers and 18 nonrisk-allele carriers, evaluated by means of a quantitative polymerase-chain-reaction analysis for all genes in the 2.5-Mb locus. The horizontal line within each box represents the median, the top and bottom of each box indicate the 75th and 25th percentile, and I bars indicate the range.

Among them, the developmental regulators IRX3 and IRX5 had genotype-associated expression, which indicated long-range (1.2-Mb) genetic control in primary preadipocytes (Figure 2C). Genotype-associated expression was not observed in whole-adipose tissue, a finding consistent with previous reports23,24; this indicated that the effect was cell type–specific and restricted to preadipocytes, which represent a minority of cells in adipose tissue (Fig. S2A in the Supplementary Appendix).


Effect of the FTO Locus on Mitochondrial Thermogenesis and Lipid Storage

To identify the biologic processes affected by altered IRX3 and IRX5expression in adipocytes, we used genomewide expression patterns in brown adipocyte–containing perirenal adipose tissue from a separate cohort of 10 nongenotyped, healthy kidney donors to identify genes with expression that was positively or negatively correlated with IRX3 and IRX5 expression. Genes that are associated with mitochondrial functions were found to have a negative correlation with IRX3 and IRX5, and genes with FXR and RXR lipid-metabolism functions were found to have a positive correlation, which suggests thatIRX3 and IRX5 may play roles in energy dissipation and storage


Figure 3A


Regulation of Obesity-Associated Cellular Phenotypes in Human Adipocytes by IRX3and IRX5., and Table S1 in the Supplementary Appendix). IRX3 and IRX5 had consistently higher mean expression in white adipose tissue from nine participants, as well as negative correlation with PGC1A and UCP1expression, as assessed with the use of interindividual expression patterns in perithyroid brown adipose tissue (Fig. S2B and S2C in the Supplementary Appendix); these findings indicated potential roles for IRX3 and IRX5 in the repression of thermogenesis.

To examine the trans-eQTL genetic control of energy balance by the FTOobesity locus, we used primary preadipocytes from risk-allele carriers and nonrisk-allele carriers to evaluate the genes with mitochondrial and FXR and RXR functions that had expression patterns most closely correlated with those of IRX3 and IRX5, as well as several known markers of energy-balance regulation (Fig. S2D and S2E in the Supplementary Appendix). As compared with nonrisk-allele carriers, risk-allele carriers had lower expression of mitochondrial, browning, and respiration genes and higher expression of lipid-storage markers, which indicated a shift from energy dissipation to energy storage.

These differences in expression were also reflected in the cellular signatures of obesity. Risk-allele carriers had increased adipocyte size, reduced mitochondrial DNA content, and a loss of UCP1 response to β-adrenergic stimulus or cold exposure (Figure 3B and 3C, and Fig. S2F in theSupplementary Appendix), as well as resistance to isoproterenol-mediated uncoupling, a decreased basal oxygen consumption rate, and a reduction in mitochondrial thermogenesis by a factor of 5 (Fig. S2G in the Supplementary Appendix); this indicated excessive accumulation of triglycerides, reduced mitochondrial oxidative capacity, reduced white adipocyte browning, and reduced thermogenesis.

Adipocyte-Autonomous Effects of IRX3 and IRX5 on Energy Balance

We next quantified the effect that manipulation of IRX3 and IRX5 expression had on thermogenesis in primary preadipocytes that were isolated from both risk-allele carriers and nonrisk-allele carriers. In preadipocytes from risk-allele carriers, IRX3 and IRX5 knockdown restored oxygen consumption and thermogenesis response to nonrisk levels, increased thermogenesis by a factor of 7 (Figure 3D), and restored UCP1 expression levels (Fig. S3A in the Supplementary Appendix). In preadipocytes from nonrisk-allele carriers, IRX3 and IRX5 overexpression reduced basal respiration and thermogenesis to risk-allele levels (with thermogenesis reduced by a factor of 8) (Figure 3D) and decreased the expression of UCP1, other regulators of mitochondrial function and thermogenesis (PGC1A, PGC1B, and PRDM16), and the β-adrenergic receptor (ADRB3), which also regulates UCP1-independent thermogenesis programs (Fig. S3B and S3C in the Supplementary Appendix). These manipulations had no significant effect on preadipocytes from participants with the reciprocal genotypes, which indicated that IRX3 and IRX5 levels recapitulate the effect that the FTO genetic variant has on thermogenesis.

To examine the organism-level effects of the repression of Irx3 in adipose tissue, we used adipose Irx3 dominant-negative (aP2-Irx3DN) mice. These mice had pronounced antiobesity characteristics, including reduced body size, body weight, fat mass, white and brown fat depots, and adipocyte size (Fig. S4A through S4G in the Supplementary Appendix). These aP2-Irx3DN mice also had resistance to weight gain on a high-fat diet, increased energy expenditure both at night and during the day, and increased oxygen consumption both at room temperature (22°C) and in thermoneutral conditions (30°C), but they did not have significant differences from control mice in food intake or locomotor activity (Fig. S4A and S4H through S4L in the Supplementary Appendix). At the molecular and cellular levels, these mice had increased mitochondrial activity and thermogenesis marker expression, reduced lipid-storage marker expression in both white and brown fat compartments, and markedly smaller adipocytes than did control mice (Fig. S4M, S4N, and S4O in the Supplementary Appendix).

Figure 4. Disruption of a Conserved ARID5B Repressor Motif by Causal SNV rs1421085 in Humans.

Panel A shows disruption of an ARID5B repressor motif in the evolutionarily conserved motif module surrounding rs1421085. The sequences shown at the top of the panel indicate the frequencies of each nucleotide, with the size scaled to indicate the information content (measured as entropy) at each position. Panel B shows adapted phylogenetic module complexity analysis (PMCA)25 scores in the FTO region for all 82 noncoding SNPs in LD (r2≥0.8) with tag SNV rs1558902, which was identified in a genomewide association study26; rs1421085 had the maximal score. Chromatin state annotation is shown for Roadmap Epigenomics reference genome E025, which corresponds to adipose-derived mesenchymal stem cells; for information on the colors used to denote chromatin states, see Figure S1A in the Supplementary Appendix. Panel C shows increased endogenous expression of IRX3 and IRX5 on single-nucleotide T-to-C editing of rs1421085 in the nonrisk haplotype of a nonrisk-allele carrier, using CRISPR–Cas9 (five clonal expansions). CRISPR–Cas9 re-editing from the engineered C risk allele back to a T nonrisk allele with the use of an alternative single guide RNA restores low endogenous IRX3 and IRX5 gene expression. Panel D shows reduced expression of IRX3 and IRX5 on C-to-T editing of the risk allele in adipocyte progenitors from a risk-allele carrier. Knockdown of ARID5B increases IRX3 and IRX5 levels, as compared….

We next evaluated the tissue-autonomous versus brain-mediated roles of Irx3 by comparing the aP2-Irx3DN mice with hypothalamus dominant-negative Ins2-Irx3DN mice.19 The aP2-Irx3DN mice had a reduction in fat-mass ratio that was 3 times as great as that in Ins2-Irx3DN mice (a reduction of 57% vs. 19%), despite the fact that transgene expression in the hypothalamus was 3 times lower than that in Ins2-Irx3DN mice (Fig. S4P and S4Q in the Supplementary Appendix), which indicated that Irx3 has a hypothalamus-independent regulatory role in whole-body energy regulation. The phenotypic effects of Irx3 repression in aP2-Irx3DN mice were also stronger than those in whole-body Irx3 knockout mice, which suggested potential dominant repressor effects in adipocytes or other tissues, and were independent of Fto gene expression, which did not change (Fig. S4P and S4R in the Supplementary Appendix).

Our findings indicate that both Irx3 and Irx5 have cell-autonomous roles: manipulation of Irx3 andIrx5 led to energy-balance differences in three mouse cellular models, including mouse embryonic fibroblast–derived adipocytes, white 3T3-L1 preadipocytes, and β-adrenergic–stimulated beige ME3 preadipocytes (Fig. S5 in the Supplementary Appendix). In each case, our results indicated that Irx3 and Irx5 induced adipocyte lipid accumulation and repressed thermogenesis in a cell-autonomous way.


Determination of the Causal Variant and Disruption of Repression by ARID5B

To predict the causal variant, the disruption of which is necessary and sufficient to cause IRX3 andIRX5 dysregulation in human preadipocytes, we used phylogenetic module complexity analysis (PMCA)25

(Figure 4A 


Disruption of a Conserved ARID5B Repressor Motif by Causal SNV rs1421085 in Humans., and Fig. S6A and S6B in the Supplementary Appendix). The highest PMCA score was found for the rs1421085 T-to-C SNV, which is in perfect linkage disequilibrium with the most significant reported SNV, rs1558902, across multiple populations (1000 Genomes Phase 1 data), a finding that is consistent with a potentially causal role.

To evaluate whether rs1421085 plays a causal role in enhancer activity, we introduced the C allele into the nonrisk haplotype in our luciferase reporter assay. The T-to-C single-nucleotide alteration increased enhancer activity levels for 10-kb and 1-kb segments centered on the variant, in both orientations and both upstream and downstream of the transcription start, which indicated a gain of enhancer activity in association with the rs1421085 risk allele (Fig. S6C and S6D in the Supplementary Appendix).

To evaluate the effect of the variant on regulator binding, we used electrophoretic mobility-shift assays (EMSAs) of adipocyte nuclear extract with probes for the risk allele and the nonrisk allele of rs1421085. We found binding for the nonrisk allele, T, which lacked enhancer activity, but no binding for the risk allele, C; this indicated that the increased enhancer activity associated with the risk allele is probably due to a loss of repressor binding rather than to a gain of activator binding (Fig. S6E in the Supplementary Appendix).

We examined disrupted motifs and regulator expression to identify potential upstream regulators. The T-to-C substitution disrupted conserved motifs for NKX6-3, LHX6, and the ARID family of regulators (Figure 4A). Among them, ARID5B had the highest expression in adipose tissue and adipocytes and was bound specifically to the nonrisk allele in EMSA competition experiments (Fig. S6E and S6F in the Supplementary Appendix). ARID5B is known to play both repressive and activating roles and was previously implicated in adipogenesis and lipid metabolism in mice.27,28. Among nonrisk-allele carriers, expression of ARID5B was negatively correlated with expression ofIRX3 and IRX5, a finding consistent with ARID5B having a repressive role. No correlation was found in risk-allele carriers, which indicates a loss of ARID5B regulation (Fig. S6G in the Supplementary Appendix).

To evaluate the causal role of ARID5B, we next examined the effects of its knockdown and overexpression on IRX3 and IRX5. ARID5B knockdown increased IRX3 and IRX5 expression in primary preadipocytes from nonrisk-allele carriers to risk-allele levels, which indicates a loss of repression, but it had no effect on preadipocytes from risk-allele carriers, which indicates epistasis with the obesity-risk haplotype (Fig. S6H in the Supplementary Appendix). Consistent with this finding, in SGBS enhancer assays, ARID5B knockdown increased the activity of preadipocytes with the nonrisk allele to risk-allele levels, which indicates a loss of repression, but had no effect on risk-allele constructs, indicating epistasis with the rs1421085 risk allele (Fig. S6I in the Supplementary Appendix). ARID5B overexpression further reduced IRX3 and IRX5 levels in nonrisk-allele carriers, which indicated that repression was strengthened, but had no significant effect on risk-allele carriers, a finding consistent with impaired ARID5B repression in association with the risk haplotype (Fig. S6J in the Supplementary Appendix).

We also evaluated the cellular effects of ARID5B-directed perturbations in primary preadipocytes from risk-allele carriers and nonrisk-allele carriers. In preadipocytes from nonrisk-allele carriers,ARID5B knockdown reduced basal oxygen consumption and lipolysis (Fig. S6K and S6L in theSupplementary Appendix) and shifted expression patterns from mitochondrial to lipid markers (Fig. S2E in the Supplementary Appendix), which indicated that ARID5B plays causal roles in energy-balance regulation. In contrast, ARID5B knockdown had no effect on preadipocytes from risk-allele carriers, a finding consistent with a loss of ARID5B control.

These results suggest that the FTO obesity variant acts through disruption of ARID5B binding in the risk haplotype, leading to a loss of repression, a gain of enhancer activity, and increases inIRX3 and IRX5 expression (Fig. S6M in the Supplementary Appendix).


C-to-T Editing of the rs1421085 Risk Variant and the Effect on Thermogenesis

Targeted genome editing technology involving CRISPR–Cas929 makes it possible to test the phenotypic effect of altering the predicted causal nucleotide rs1421085 in its endogenous genomic context, in isolation from the other obesity-associated genetic variants in the same haplotype. We used CRISPR–Cas9 in primary preadipocytes with two separate guide RNAs, one for rs1421085 C-to-T rescue of the ARID5B motif disruption in risk-allele carriers and one for rs1421085 T-to-C disruption of the ARID5B motif in nonrisk-allele carriers.

We first evaluated the effect of rs1421085 editing on IRX3 and IRX5 expression levels. Starting from preadipocytes of a nonrisk-allele carrier, T-to-C editing doubled endogenous IRX3 and IRX5expression, to levels seen in risk-allele carriers; starting from the edited preadipocytes, C-to-T re-editing back to the nonrisk allele restored low expression levels (Figure 4C). Starting from the risk haplotype, C-to-T editing reduced IRX3 and IRX5 to nonrisk-allele levels, but only in the presence of ARID5B (Figure 4D); this established that disruption of ARID5B repression by rs1421085 is the mechanistic basis of the IRX3 and IRX5 dysregulatory event that mediates the effects of the FTOlocus on obesity.

Next, we evaluated the role of rs1421085 editing during differentiation of white and beige adipocytes, by studying differences in expression between edited and unedited preadipocytes during differentiation. Unedited adipocytes from a risk-allele carrier had a peak in IRX3 and IRX5expression during days 0 and 2 of preadipocyte differentiation into adipocytes; expression during early differentiation was reduced to nonrisk-allele levels by rs1421085 editing, which indicated a causal role of rs1421085 in developmental gene expression programs.

(Figure 5A


Rescue of Metabolic Effects on Adipocyte Thermogenesis through Editing of SNV rs1421085 in a Risk-Allele Carrier. The causal role of rs1421085 was further reflected in a significant increase in the expression of thermogenesis regulators (ADRB3, DIO2, PGC1A, and UCP1) and mitochondrial markers (NDUFA10, COX7A, and CPT1) in differentiating preadipocytes (Figure 5B), which indicated that C-to-T editing of the risk allele rescued thermogenesis regulatory programs.

Last, we evaluated the role of rs1421085 editing in cellular signatures of obesity by quantifying phenotypic differences between edited and unedited adipocytes. A causal role in the regulation of energy balance was indicated by the fact that C-to-T rescue of rs1421085 in edited adipocytes resulted in a reduction in gene expression for lipid storage and lipolytic markers (Fig. S2E and S8A in the Supplementary Appendix), an increase by a factor of 4 in basal metabolic rate and β-adrenergic oxygen consumption, and an increase by a factor of 7 in thermogenesis (Figure 5C, and Fig. S7B in the Supplementary Appendix). In particular, rescue of the ARID5B motif in C-to-T edited preadipocytes restored the strong dependence of mitochondrial respiration on ARID5B that is seen in nonrisk-allele carriers (Fig. S7C in the Supplementary Appendix).

These results indicate that the rs1421085 T-to-C single-nucleotide alteration underlies the association between FTO and obesity by disrupting ARID5B-mediated repression of IRX3 andIRX5. This disruption leads to a developmental shift from browning to whitening programs and loss of mitochondrial thermogenesis (Figure 5D).


Our work elucidates a potential mechanistic basis for the genetic association between FTO and obesity and indicates that the causal variant rs1421085 can disrupt ARID5B repressor binding; this disruption results in derepression of IRX3 and IRX5 during early adipocyte differentiation. This process could lead to a cell-autonomous shift from white adipocyte browning and thermogenesis to lipid storage, increased fat stores, and body-weight gain.

To translate the results of genomewide association studies into mechanistic insights, we combined public resources (epigenomic annotations, chromosome conformation, and regulatory motif conservation), targeted experiments for risk and nonrisk haplotypes (enhancer tiling, gene expression, and cellular profiling), and directed perturbations in human primary cells and mouse models (regulator–target knockdown and overexpression and CRISPR–Cas9 genome editing). These methods are specific to the elucidation of noncoding variants, which constitute the majority of signals in genomewide association studies; 80% of the trait-associated loci identified in such studies lack protein-altering variants, and 93% of the top hits are noncoding.30

The FTO association with obesity is unusual in many ways. First, rs1421085 has both a high frequency and a strong effect size,31 which suggests positive selection or bottlenecks (e.g., 44% frequency in European populations vs. 5% in African populations). Second, rs1421085 has switchlike behavior in enhancer activity, target-gene expression, and cellular phenotypes, possibly because of selective pressures on energy-balance control for rapid adaptation. Third, rs1421085 acts specifically in the early differentiation of preadipocytes, which emphasizes the importance of profiling diverse tissues, cell types, and developmental stages. Fourth, enhancer activity is found only for the risk allele, which emphasizes the importance of profiling both alleles. Finally, rs1421085 leads to a gain of function (increased enhancer, IRX3, and IRX5 activity); this is a rare property in protein-coding variants but may be common in noncoding variants.

The apparent genetic link between obesity and cell-autonomous adipocyte browning suggests a central role of beige adipocyte thermogenesis in whole-body energy metabolism in humans, a role that is consistent with that suggested in recent reports on PRDM16 in mice.9 IRX3 and IRX5 have evolutionarily conserved roles, and the ARID5B motif lies in a module that is functionally conserved across multiple mammalian species; this indicates that adaptive thermogenesis circuits are conserved, and IRX3 and IRX5 probably play both UCP1-dependent and UCP1-independent roles. Even though IRX3 and IRX5 dysregulation by rs1421085 was restricted to early differentiation, their effects persisted in mature adipocytes, and the targeting of these genes can have broader effects.

Last, we found that direct manipulation of the ARID5B–rs1421085–IRX3/IRX5 regulatory axis in primary cell cultures of adipocytes from patients reversed the signatures of obesity. This indicates that in addition to changes in physical activity and nutrition, manipulation of mitochondrial thermogenesis26 offers a potential third pathway for shifting between energy storage and expenditure in a brain-independent and tissue-autonomous way in humans.

In summary, our work elucidates a mechanistic basis for the strongest genetic association with obesity. Our results indicate that the SNV rs1421085 underlies the genetic association between theFTO locus and obesity. The SNV disrupts an evolutionarily conserved motif for the ARID5B repressor, which leads to loss of binding, derepression of a potent preadipocyte superenhancer, and activation of downstream targets IRX3 and IRX5 during early differentiation of mesenchymal progenitors into adipocyte subtypes. This results in a cell-autonomous shift from white adipocyte browning to lipid-storage gene expression programs and to repression of basal mitochondrial respiration, a decrease in thermogenesis in response to stimulus, and an increase in adipocyte size. Manipulation of the uncovered pathway, including knockdown or overexpression of the upstream regulator ARID5B, genome editing of the predicted causal variant rs1421085, and knockdown or overexpression of target genes IRX3 and IRX5, had a significant effect on obesity phenotypes.


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Targeting of GLUT1 Glucose Transporter

Larry H. Bernstein, MD, FCAP, Curator



Prolactin-induced Subcellular Targeting of GLUT1 Glucose Transporter in Living Mammary Epithelial Cells

Arieh Riskin, M.D., M.H.A., and Yehudit Mond, M.Sc.
Rambam Maimonides Med J  Oct 2015; 6( 4): e003

Abbreviations: ECFP, enhanced cyan fluorescent protein; EGFP, enhanced GFP; GFP, green fluorescent protein; GM, growth medium; HMEC, human mammary epithelial cells; MEC, mammary epithelial cells; MMEC, mouse mammary epithelial cells; pECFP, plasmid vector ECFP; pEGFP, plasmid vector EGFP; PCR, polymerase chain reaction; SM, secretion medium. Citation: Riskin A, Mond Y. Prolactin-induced Subcellular Targeting of GLUT1 Glucose Transporter in Living Mammary Epithelial Cells. Rambam Maimonides Med J 2015;6 (4):e0038.

Background: Studying the biological pathways involved in mammalian milk production during lactation could have many clinical implications. The mammary gland is unique in its requirement for transport of free glucose into the cell for the synthesis of lactose, the primary carbohydrate in milk. Objective: To study GLUT1 trafficking and subcellular targeting in living mammary epithelial cells (MEC) in culture.

Methods:Immunocytochemistry was used to study GLUT1 hormonally regulated subcellular targeting in human MEC (HMEC). To study GLUT1 targeting and recycling in living mouse MEC (MMEC) in culture, we constructed fusion proteins of GLUT1 and green fluorescent protein (GFP) and expressed them in CIT3 MMEC. Cells were maintained in growth medium (GM), or exposed to secretion medium (SM), containing prolactin.

Results: GLUT1 in HMEC localized primarily to the plasma membrane in GM. After exposure to prolactin for 4 days, GLUT1 was targeted intracellularly and demonstrated a perinuclear distribution, co-localizing with lactose synthetase. The dynamic trafficking of GFP-GLUT1 fusion proteins in CIT3 MMEC suggested a basal constitutive GLUT1 recycling pathway between an intracellular pool and the cell surface that targets most GLUT1 to the plasma membrane in GM. Upon exposure to prolactin in SM, GLUT1 was specifically targeted intracellularly within 90–110 minutes.

Conclusions: Our studies suggest intracellular targeting of GLUT1 to the central vesicular transport system upon exposure to prolactin. The existence of a dynamic prolactin-induced sorting machinery for GLUT1 could be important for transport of free glucose into the Golgi for lactose synthesis during lactation.

KEY WORDS: CIT3 mouse mammary epithelial cells, green fluorescent protein, GLUT1 glucose transporter, human mammary epithelial cells, prolactin


Biology of Milk Production and Transport Pathways of Milk Constituents Females of all mammalians bear mammary glands, and milk secretion and lactation is a characteristic feature of all mammalian species, which are the only organisms that produce copious glandular skin secretions to feed their young.1,2 Lactation is a highly complex and evolutionarily ancient strategy of all mammals, providing their offspring with a highly digestible, concentrated, nutritionally balanced diet, while allowing adult mammals to evolve a wide range of developmental and reproductive strategies and specialize on diets that could either be too difficult to capture or digest or would be insufficient to cover the high nutritional needs of their small rapidly growing offspring.1–3 Lactation helps mammalian mothers cope with unreliable food supplies, because lactating females can draw on their nutrient reserves for milk production, suggesting an evolutionary advantage for their dependent offspring, since milk intake promotes growth, fitness, and survival of the young.2,4,5 Beyond nourishment of the neonate, milk also helps establish immunological and endocrine competence in the offspring. Milk’s nutrient composition varies extensively across mammalian species, as a function of evolutionary history, maternal nutrient intake, duration of milk production, and stage of lactation.2,5 Milk is a complex mixture whose composition reflects different transport and secretion mechanisms within the mammary gland that aim to answer the different nutritional needs of mammalian neonates.6

The lactating mammary gland is composed of branching ducts ending in alveolar clusters where milk is produced. A single layer of polarized secretory epithelial cells forms the alveolar wall. The alveoli are surrounded by myoepithelial cells and are embedded in vascularized connective tissue stroma. While growth of the mammary gland and secretion of milk are stimulated by growth hormone, prolactin, adrenocortical steroids, estrogens, and progesterone, ejection of milk requires contraction of myoepithelial cells stimulated by oxytocin.6,7 The cytoplasm of the secretory alveolar epithelial cells is filled with numerous mitochondria, extensive rough endoplasmic reticulum network, well-developed Golgi apparatus, and secretory vesicles in the apical region of the cell adjacent to the alveolar lumen. The basal side of the alveolar epithelial cells lies on a basement membrane that separates them from the stroma and vascular system. In between, epithelial cells are connected to each other by an apical complex of tight junctions that inhibit direct paracellular exchange of substances between the vascular compartment and milk in the alveolar lumen. There are five pathways by which solutes, including proteins, lipids, ions, nutrients, and water, can be transported into the milk. Four are transcellular, involving transport across at least two membrane barriers, while the fifth is para-cellular and allows direct exchange of interstitial and milk components: (1) The exocytic pathway is for endogenously generated aqueous soluble substances, including lactose, oligosaccharides, the major milk proteins, citrate, phosphate, calcium, and other nutrients, which is similar to exocytic pathways in other cell types; (2) The transport pathway for milk lipids is unique to the mammary gland generating the milk fat globules; milk lipids, primarily triacylglycerides, are synthesized in the smooth endoplasmic reticulum in the basal region of the lactating alveolar cell—the newly synthesized lipid molecules are coated by protein membranes to form small storage structures called cytoplasmic lipid droplets that are transported to the apical plasma membrane, where they are secreted by a unique budding process as membrane-enveloped structures, i.e. the milk fat globules; (3) The trans-cytosis pathway transports macromolecules derived from the serum or stromal cells, including serum proteins (such as immunoglobulin, albumin, and transferrin), hormones (such as insulin, prolactin, and estrogen), and stromalderived substances (such as secretory immunoglobulin A, cytokines, and lipoprotein lipase); (4) Various membrane transport pathways transport ions and small molecules (such as glucose, amino acids, and water) across the basal and apical plasma membranes of the polarized alveolar epithelial cell; and (5) The para-cellular pathway provides a direct route for transfer of serum and interstitial substances into the milk. These transport pathways are regulated by the functional stage of the mammary gland and by hormones and growth factors.6

Studying mammary gland biology, transport pathways of milk constituents, and their influence on milk production in general, and specifically investigating glucose transport mechanisms in mammary epithelial cells (MEC) that could influence lactose synthesis and thus milk volume, is important and could have many clinical implications, including in breastfeeding support to mothers, in the dairy industry, and in oncology. Breast cancer tumor cells may utilize some of the MEC transport pathways to support metabolically their rapid uncontrolled growth.6,8

Glucose Transport Pathways in the Mammary Epithelial Cells Human milk is rich in lactose, which is the major osmotic constituent of human milk and thus the major determinant of milk volume.9 Lactose is a disaccharide composed of glucose and galactose. Lactose is found only in milk and is the primary carbohydrate in milk. The final step in the biosynthesis of lactose from UDP-galactose and glucose is catalyzed by lactose synthetase, a complex of - lactalbumin and the Golgi enzyme 1,4-galactosyltransferase.10 1,4-Galactosyltransferase is embedded in the inner surface of Golgi membranes. It is membrane-bound and directed towards the lumen of the cisternal space.10,11 Galactosyltransferase is found in most tissues and is involved in protein glycosylation. In mammals 1,4-galactosyltransferase has been recruited for a second biosynthetic function, the production of lactose. This function takes place exclusively in the lactating mammary gland. Galactosyltransferase has a relatively poor affinity for glucose (Km~1 M). The affinity of this enzyme is profoundly modified by transient association with -lactalbumin, which creates a binding site for glucose, so that the affinity of the transferase for glucose increases about 500-fold (Km~2 mM).10 This allows the synthesis of lactose in the Golgi to occur at the physiological concentrations found in the mammary cell. -Lactalbumin is a milk whey protein that is not catalytically active by itself, but is necessary for the synthesis of lactose. The initiation of -lactalbumin synthesis that occurs at parturition is required for the initiation of copious milk production, but is neither the only factor nor the limiting factor controlling lactose synthesis.11,12 Availability of glucose and UDP-galactose to the lactose synthase enzyme complex in the Golgi apparatus may be rate-limiting for lactose synthesis. Many cells possess active mechanisms for the uptake of nucleotide sugars such as UDP-galactose into the Golgi, which are essential to protein glycosylation. In contrast, the mammary gland is unique in its need to transport free glucose into the Golgi. The main known isoform of glucose transporters expressed in the mammary gland is GLUT1 (SLC2A1).9,13,14–20 Levels of GLUT1 increase progressively during pregnancy, reaching their highest levels during lactation,15,16 and this is dependent on prolactin.21 In most cells GLUT1 normally resides in the plasma membrane and is responsible for basal glucose uptake. In polarized epithelial cells, including mammary cells, GLUT1 is targeted primarily to the basolateral membrane.22 The role of GLUT1 in glucose transport into Golgi has been controversial.15,23,24 Evidence from in vivo and in vitro studies demonstrated unique hormonally regulated intracellular targeting of GLUT1 from the plasma membrane to a low-density intracellular compartment in mouse mammary gland during lactation.16,23–25 Further work distinguished this compartment from Golgi, suggesting that the hormonally induced intracellular targeting of GLUT1 in lactating MEC is into a Brefeldin A-sensitive low-density vesicle that may represent a subcompartment of cisGolgi.25 This hormonally regulated subcellular targeting of GLUT1 may have an important role for lactose synthesis in MEC during lactation.

The aim of this work was to study GLUT1 subcellular targeting in living human and mouse mammary epithelial cells (HMEC and MMEC) in culture; and to study GLUT1 intracellular trafficking in living MMEC in culture under the effects of the lactogenic hormones that regulate it. Our hypothesis was that there would be dynamic basal trafficking of GLUT1 in living MEC that would target most GLUT1 to the basolateral membrane under maintenance conditions, and intracellularly upon exposure to prolactin, which should be the main hormonal stimulus that drives this translocation during lactation. Our studies could then complete the previous in vitro findings in fixed cell25 by adding the dynamic observations in living MEC that could in turn relate to the in vivo findings.16

GLUT1 Subcellular Targeting in HMEC Immunocytochemistry of HMEC in maintenance MEGM medium using highly specific anti-GLUT1 antibody demonstrated plasma membrane distribution of GLUT1 as well as an intracellular, mostly perinuclear, pattern (Figure 1A). After exposure to the prolactin-rich MESM for 4 days, GLUT1 was specifically targeted intracellularly, demonstrating a perinuclear pattern. A distinct nuclear membrane distribution of GLUT1 was also observed under these conditions (Figure 1B). In secretion medium GLUT1 green signal colocalized with the blue signal of pECFP-Golgi (Figure 2A, B, C). It also co-localized with the red signal of alpha-lactalbumin and alpha-mannosidase II (Figure 2D, E, F, and G, H, I, respectively). Partial co-localization was demonstrated with the red signal of beta-COP and with transferrin-Texas Red (Figure 3A, B, C, and D, E, F, respectively). No co-localization was demonstrated after staining the cells with the red stain, BODIPY-TR ceramide (Figure 4A, B, C, D, E, F). GLUT1 Fusion Chimeras to EGFP Exhibit Normal GLUT1 Targeting in Vitro We subcloned GLUT1 cDNA into pEGFP to create N- and C-terminus fusions. The recombinant plasmid vectors were introduced into CIT3 cells by transient liposome-mediated transfection, achieving fluorescent expression in 20%–30% of the cells.

Figure 1. Exposure to Prolactin Causes Intracellular Targeting of GLUT1. Cells were fixed and exposed to specific anti-GLUT1 primary antibody. Bar 15 m. A: In maintenance medium, GLUT1 demonstrates primarily a plasma membrane distribution as well as some intracellular mostly perinuclear staining. B: After exposure to prolactin-rich medium for 4 days, GLUT1 was specifically targeted intracellularly, demonstrating a perinuclear pattern, as well as a distinct nuclear membrane staining.

Figure 2. After Exposure to Prolactin, GLUT1 Colocalizes with ECFP-Golgi, alpha-Lactalbumin and alpha- Mannosidase II. Fluorescent images were captured 60 hours after transfection with 1 microgram of pECFP-Golgi. Cells were maintained in prolactin-rich medium for 4 days, before they were fixed and stained with specific anti-GLUT1, anti-alpha- lactalbumin or anti-alpha-mannosidase II. GLUT1 is shown in green, and alpha-lactalbumin or alpha-mannosidase II in red after staining with FITC-conjugated and Texas Redconjugated secondary antibodies, respectively. ECFPGolgi emits cyan-blue fluorescence when exposed to fluorescent light at the appropriate wavelength. Bar 10 microm. A, D, G: GLUT1 signal. B: ECFP-Golgi signal. E, H: alpha-Lactalbumin and alpha-mannosidase II signals, respectively. C, F, I: Superimposed images. Perinuclear colocalization of GLUT1 and ECFP-Golgi is shown as areas of coincident staining (C). Co-localization of GLUT1 and alpha-lactalbumin or alpha-mannosidase II appear as areas of coincident staining, giving rise to yellow signal (F, I).

Figure 3. After Exposure to Prolactin, GLUT1 only Partially Co-localizes with beta-COP and TransferrinTexas Red in Endosomes. Cells were maintained in prolactin-rich medium for 4 days, before they were fixed and stained with specific anti-GLUT1 or anti-beta-COP primary antibodies. Some cells were exposed shortly to transferrin-Texas Red staining before fixation and exposure to anti-GLUT1. GLUT1 is shown in green, and beta-COP in red after staining with FITC-conjugated and Texas Redconjugated secondary antibodies, respectively. Transferrin stain appears in red. A, D: GLUT1 signal. B, E: beta-COP and transferrin (short-term exposure) signals, respectively. C, F: Superimposed images. Partial co-localization of GLUT1 with beta-COP or transferrin in endosomes appears as areas of coincident staining, giving rise to yellow signal.

Figure 4. After Exposure to Prolactin, GLUT1 Does not Co-localize with BODIPY-TR Ceramide. Cells were maintained in prolactin-rich medium for 4 days, before they were fixed and stained with specific anti-GLUT1 primary antibody, or stained with BODIPYTR ceramide. GLUT1 is shown in green after staining with FITC-conjugated secondary antibody. BODIPY-TR ceramide appears in red. A, D: GLUT1 signal. B, E: BODIPY-TR ceramide signal. C, F: Superimposed image. There is little overlap of GLUT1 green signal with BODIPY-TR ceramide.

GLUT1 Fusion Chimeras to EGFP Exhibit Normal GLUT1 Targeting in Vitro We subcloned GLUT1 cDNA into pEGFP to create N- and C-terminus fusions. The recombinant plasmid vectors were introduced into CIT3 cells by transient liposome-mediated transfection, achieving fluorescent expression in 20%–30% of the cells.

Immunocytochemistry studies with antibodies against the C-terminus of native GLUT1 showed that the fluorescent signal of both GLUT1 chimeras to GFP co-localized extensively with native GLUT1 (Figure 5). This result validated the use of the GLUT1-GFP fusion proteins to study dynamic aspects of GLUT1 targeting.

Figure 5. GLUT1 Chimeras to GFP Co-localize with Native GLUT1 in CIT3 Cells in SM. EGFP-GLUT1 fusion protein (B) exhibits the same intracellular distribution as native GLUT1 (A). Superimposed images (C) demonstrate that co-localization of native GLUT1 and EGFP-GLUT1 fusion protein appears as areas of coincident staining, giving rise to yellow signal. GLUT1-EGFP fusion protein (E) exhibits the same intracellular distribution as native GLUT1 (D). Superimposed images (F) demonstrate that co-localization of native GLUT1 and GLUT1-EGFP fusion protein appears as areas of coincident staining, giving rise to yellow signal.

EGFP chimeras to GLUT1 demonstrated change in subcellular distribution, after 96 hours of exposure to SM. In GM the fusion proteins were targeted mainly to the basolateral plasma membrane. In SM GLUT1-EGFP chimeras were mostly targeted into the cell, exhibiting unique perinuclear distribution with punctate pattern scattered through the cytoplasm (Figures 5 and 6). Both the N- and C-terminus fusions to GLUT1 (EGFP-GLUT1 and GLUT1-EGFP, respectively) exhibited the same targeting patterns (Figure 6).

Figure 6. CIT3 Cells Expressing EGFP Fusion to the N- and C-termini of GLUT1 in GM and after 4 Days in SM. High-power images. Upper left: Plasma membrane targeting of EGFP-GLUT1 in GM. Lower left: Intracellular pattern of EGFP-GLUT1 signal in differentiated cells in SM. Upper right: Plasma membrane targeting of GLUT1-EGFP in GM. Lower right: Intracellular targeting of GLUT1-EGFP with perinuclear punctate distribution in cells exposed to SM. All images captured with 0.25 s exposure time and are at 40× magnification, except for the upper left image, which was optimized at 0.5 s exposure time and higher power (60×).

Degree of Differentiation Affects Intracellular Targeting of GLUT1 Static images of cells at different levels of differentiation in SM demonstrated that the degree of differentiation affected the level and distribution of GLUT1 targeting in perinuclear localization (Figure 7). In GM the GLUT1-EGFP signal was mostly targeted to the plasma membrane (Figure 7A). In SM GLUT1-EGFP fusion proteins exhibited the unique perinuclear punctate pattern, described above (Figure 7B). However, in the more differentiated MEC in SM, where vesicles and fat globules were morphologically prominent, the GLUT1-EGFP signal was no longer punctate, but targeted to the boundaries of these vesicles, still maintaining mostly perinuclear distribution. There was signal throughout the cytoplasm as well (Figure 7C, D). This differentiation-related GLUT1 subcellular targeting may also support the assumption that dynamic transport systems are involved in the trafficking of the GFP-GLUT1 fusion proteins, as will be shown below.

Figure 7. Degree of Differentiation Affects Intracellular Targeting of GLUT1. Static images of CIT3 cells in GM and at different levels of differentiation in SM. Panel A: In GM; B, C, D: In SM at different levels of differentiation. The upper left figure of each panel is GLUT1-EGFP signal. The upper right figure is staining of the same cells with BODIPY-TR ceramide (Molecular Probes, Inc., Eugene, OR, USA), which is a dye that marks the trans-Golgi in red. Living CIT3 cells were pre-incubated with 5 nmol/mL of BODIPY-TR ceramide at 4C for 30 minutes. Lower left figure is superimposed image of the upper figures. Lower right figure is phase contrast image of the cells to show the different levels of differentiation.

GLUT1 Intracellular Trafficking in Secretion Media Seems to be Dynamic Living CIT3 cells transfected with GLUT1-EGFP and kept in SM were followed by time-lapse imaging, where trafficking of GLUT1 fusion proteins could be seen (Figure 8; also available online as a supplemental YouTube video clip). This trafficking seems to be dynamic and could suggest that transport systems are possibly involved in it.

Figure 8. GLUT1 Intracellular Trafficking in SM is Dynamic. Figure 8 is also available online as a YouTube video clip at: The figure plates are from frames 8 minutes apart. All images captured with 0.25 s exposure time and are at 40× magnification.

Secretion Medium Induces GLUT1 Intracellular Targeting in Living Mammary Cells When living CIT3 cells, transfected with GLUT1- EGFP and kept in GM, were exposed to SM, dynamic trafficking of GLUT1 fusion proteins was demonstrated intracellularly, starting after approximately 50–60 minutes, with maximal intracellular targeting within 90–110 minutes. When the cells were returned to GM, most of the changes were reversible within 1–2 hours, although not fully, with redistribution of the fluorescent GLUT1 chimera mostly in the plasma membrane (Figure 9; also available online as a supplemental YouTube video clip).

Figure 9. Lactogenic Hormones in SM Induce GLUT1 Intracellular Targeting in Living Mammary Cells. Figure 9 is also available online as a YouTube video clip at: The figure plates are from frames 16 minutes apart. All images captured with 0.5 s exposure time and are at 40× magnification.

Prolactin Induces GLUT1 Intracellular Targeting in Living Mammary Cells Exposure of CIT3 cells kept in GM to SM containing prolactin without hydrocortisone caused the same changes in GLUT1 subcellular targeting as were seen with full SM. Dynamic trafficking of GLUT1 fusion proteins intracellularly was demonstrated, starting after approximately 50–60 minutes, with maximal intracellular targeting within 90–110 minutes. When the cells were returned to GM, most of the changes were reversible within 1–2 hours, although not fully. The same response was reproduced with different prolactin concentrations (300 ng/mL, 30 ng/mL), as low as 3 ng/mL (compared to the 3 microg/mL of prolactin usually used in SM). Representative results after exposure of CIT3 cells kept in GM to 30 ng/mL prolactin are shown (Figure 10; also available online as a supplemental YouTube video clip). We were not able to demonstrate doseresponse relations with the different prolactin concentrations, possibly because the results are qualitative, rather than quantitative. There was no difference in the time required to achieve maximal effect with different prolactin concentrations. Secretion medium containing hydrocortisone (3 microg/mL as in full SM) without any prolactin caused no change in GLUT1 subcellular distribution.

Figure 10. Prolactin Induces GLUT1 Intracellular Targeting in Living Mammary Cells. The response here was reproduced with prolactin concentrations of 30 ng/mL. Figure 10 is also available online as a YouTube video clip at: The figure plates are from frames 20 minutes apart. All images captured with 2 s exposure time and are at 60× magnification.

DISCUSSION Human milk contains about three times more lactose than does rodent milk, suggesting that glucose transport mechanisms can be very important in humans. Thus, before studying GLUT1 subcellular trafficking in a CIT3 MMEC model, we first examined whether hormonally regulated subcellular targeting of GLUT1 occurs in HMEC upon conditions mimicking lactation. For this, we utilized immunofluorescent staining of HMEC to demonstrate prolactin-dependent co-localization of GLUT1 with several Golgi markers.

The distribution of GLUT1, as demonstrated using immunocytochemistry with C-terminusspecific anti-GLUT1 antibody, was primarily in plasma membrane, as well as some intracellular perinuclear punctate pattern, when the cells were maintained in baseline growth medium. Upon exposure to prolactin in secretion media, GLUT1 specifically redistributed from the plasma membrane, demonstrating a perinuclear distribution with a pattern that may represent the Golgi or related structures in the central intracellular vacuolar trafficking system. A distinct perinuclear membrane distribution of GLUT1 was also observed under these conditions. Co-localization studies gave insight as to the possible Golgi subcompartment that GLUT1 resides in. GLUT1 was targeted intracellularly, co-localizing with components of lactose synthetase complex. It co-localized with ECFP-Golgi, the cyan fluorescent protein fused to the membrane-anchoring signal specific to beta 1,4-galactosyltransferase, identifying the medial/trans region of the Golgi. This colocalization gave another spatial support to its role in the transport of free glucose for lactose synthesis by beta 1,4-galactosyltransferase in the inner cisternae of the Golgi apparatus. This was further supported by the co-localization of GLUT1 with alpha-lactalbumin, which is the milk whey protein that is not catalytically active, by itself, but which in association with beta 1,4-galactosyltransferase is necessary for the synthesis of lactose.53 The initiation of alpha-lactalbumin synthesis that occurs at parturition is required for the initiation of copious milk production, but is neither the only factor nor the limiting factor controlling lactose synthesis.54 GLUT1 also colocalized with the medial-Golgi marker, alpha- mannosidase II; however, it did not co-localize with the trans-Golgi marker, BODIPY-TR ceramide. Only partial co-localization was demonstrated with beta- COP, which is a cis-Golgi marker.

The co-localization studies suggest intracellular targeting to the central vesicular transport system, which may represent a cis/medial-Golgi subcompartment. These findings are in agreement with previous findings in the mouse mammary gland16 and in CIT3 MMEC.25,55 Partial co-localization was also noted with transferrin-Texas Red after brief exposure, which marks the endosomes. The possible identification of GLUT1 in endosomes suggests that GLUT1 sorting is a continuous, dynamic process.55 Further work delineating the molecular mechanism of GLUT1 sorting and the targeting determinants it recognizes should improve our understanding of a key regulatory step of milk production in the nursing mother.

Apparent nuclear membrane staining for GLUT1 seen in HMEC has not been reported previously in any cell type, and its significance is a matter for speculation and further study. There is evidence to suggest that the apical nuclear envelope may serve as an intermediary connection between the endoplasmic reticulum and the Golgi.56 Also, the perinuclear staining of GLUT1 noted may actually be one of the perinuclear endosomal recycling compartments.52

Our findings in HMEC illustrate a potential mechanism for the delivery of free glucose to the Golgi, in what may be the rate-limiting step for lactose synthesis and milk production. In addition to its widely recognized role in the uptake of glucose by cells, GLUT1 may also mediate glucose transport between intracellular compartments.

The in vitro study of a dynamic process required developing a system of living cells with labeling of GLUT1. Green fluorescent protein is a reporter molecule for monitoring gene expression and protein localization in vivo, in situ, and in real time.57–63 Green fluorescent protein is expressed in eukaryotic cells as a fusion protein that serves as a “fluorescent tag.” The use of fluorescent fusion proteins of GLUT1 allows the study of the same cells over time, permitting studies of exocytosis and endocytosis, not just steady-state distributions. Using GFP fusion to GLUT1 is ideal for studying intracellular trafficking and subcellular targeting of GLUT1 in MEC under hormonal stimulation. It also permits evaluation of chimeric protein targeting in an antibody-independent fashion and confirms that we are studying GLUT1 and not a novel, lactation specific glucose transporter isoform that shares the GLUT1 epitope. The GLUT1 cDNA sequence29 was subcloned into pEGFP and introduced into CIT3 cells by transient transfection, with over-expression of the fluorescent GLUT1 in approximately a quarter of the cells. The intracellular targeting of GLUT1- EGFP chimera was consistent from cell to cell.

Lactogenic hormones in SM changed subcellular targeting of GFP fusion chimeras to GLUT1 from a plasma membrane distribution to an intracellular pattern, predominantly perinuclear and punctate, but also throughout the cytoplasm. The fact that this pattern was consistent with the distribution of native GLUT1 supported the use of GLUT1 chimeras to GFP as a model for studying GLUT1 intracellular targeting in MEC. Since the behavior and intracellular distribution of both the N- and C-fusion chimeras of GLUT1 to GFP were consistently the same, further studies were carried out with only one of them (GLUT1-EGFP). The level of differentiation of the lactating MEC in SM affected the degree of GLUT1 intracellular targeting and the distribution of its cytoplasmic, mostly perinuclear, localization. This fits well the previous in vivo and in vitro findings16,25 and actually also points to the possible involvement of intracellular membrane vesicular trafficking systems in GLUT1 intracellular targeting.

Living mouse MEC kept in SM demonstrated dynamic trafficking of GLUT1-EGFP fusion proteins. Careful tracking of these fluorescent GLUT1 vesicles excluded random movement and actually suggested that the dynamic intracellular targeting of GLUT1 may be mediated through altering GLUT1 exocytosis and endocytosis.

When CIT3 cells kept in GM were exposed to SM, the changes in GLUT1 targeting from mostly a plasma membrane pattern to an intracellular pattern occurred within 60–120 minutes. The maximal intracellular translocation of GLUT1-EGFP green fluorescent signal after exposure to SM was noted at 100–110 minutes. Some of this effect was reversible within 60–120 minutes upon withdrawal of SM, but we were not able to demonstrate full reversibility of the process in our in vitro system. The relatively rapid changes in GLUT1 targeting in living MMEC exposed to SM, which took place within minutes to hours, were in accordance with the findings from the in vivo studies of forced weaning, demonstrating reversible changes in GLUT1 subcellular targeting within 3–5 hours.16 These findings are also supported by the previous observation that, as early as 15 minutes after exposure of mammary tissue fragments from lactating rabbits to prolactin, the cell morphology already changed with marked increase in the relative volume occupied by the Golgi region.64

The next step was to define which of the hormones in SM is responsible for GLUT1 intracellular targeting. Exposure of CIT3 cells kept in GM to SM containing prolactin without hydrocortisone caused the same changes in GLUT1 subcellular targeting as seen with full SM. The same response was reproduced with prolactin concentrations as low as 3 ng/mL (compared to the 3 g/mL of prolactin usually used in SM). The serum concentration range of prolactin in lactating mothers is 20–300 ng/mL.65 However, we were not able to demonstrate dose-response effects with the different prolactin concentrations. The GLUT1-EGFP intracellular signal translocation took place at approximately the same time (100–120 minutes) with 3 ng/mL prolactin as it did with 3 g/mL of prolactin in the full SM. Further studies are needed to demonstrate dosedependent effects of prolactin, expressed as different levels of intracellular green fluorescent signal of GLUT1 chimeras, but this requires a more quantitative recording of the signal that unfortunately we did not have in these studies. Secretion medium containing hydrocortisone without any prolactin caused no change in GLUT1 subcellular distribution, thus excluding it as a cause for GLUT1 intracellular targeting in SM. Further studies are also needed to explore the effects of prolactin combined with other hormones, such as estrogen.

The suggestion that GLUT1 does not solely act at the plasma membrane, but may function in an intracellular organelle as well, conceptually complements the well-known insulin-regulated targeting of GLUT4,66 and to a lesser extent of GLUT1,38 to their site of action, the plasma membrane, in fat and muscle cells. Our results suggest the existence of a prolactin-induced, cell type-specific, developmental stage-specific sorting machinery for GLUT1 in MEC, and supports glucose transport as a potential ratelimiting step for lactose synthesis during lactation. The ability of the system to respond quickly to hormonal changes by altering the transport, and thus the availability of free glucose for lactose synthesis, is complementary to the well-known insulin-regulated targeting of GLUT4 to the plasma membrane in fat and muscle cells, where GLUT4 is available for glucose uptake into the cell within minutes.67 This machinery offers another level of regulation of lactose synthesis by altering GLUT1 targeting within minutes to hours, as was demonstrated also in vivo. 16 This step may not require new protein synthesis, or increase in the total amount of GLUT1 or enzymes involved in lactose synthesis, which takes longer. Our study relied only on immuno-histochemistry analysis, and further studies including Western blot and real-time PCR are needed to address the possibility of up- or downregulation of GLUT1.

Despite the fact that in our first experiments we have demonstrated specific intracellular targeting of GLUT1 from the plasma membrane of HMEC upon exposure to prolactin, the main limitation of this part of the dynamic studies is that it was limited to MMEC, and more specifically to CIT3 cell lines. Thus, these conclusions cannot be currently generalized or extended to other mammals, including humans. The suggestion that glucose transporters, other than GLUT1, may be involved in glucose regulation in MEC during lactation,18,68–71 and that their role may be more significant in other mammals, and influenced by factors other than lactogenic hormones,72–75 needs to be addressed.

Another limitation of this study is the issue of cell-to-cell variability in the phenotype of mouse MEC in the culture. Cell phenotype could vary in many ways, including morphology, intracellular lipid deposition, apparent states of differentiation, and GLUT1 targeting kinetics. This is an inherent limitation of a descriptive study based on microscopic findings. To decrease a possible selection bias of the cells we were studying, we tried to select representative cells on a lower-power field before studying them in a high-power field. We also repeated each experiment more than three times, and verified that the results were reproducible. Yet, such selection bias cannot be fully excluded.

A further limitation of our descriptive colocalization studies is the use of fluorescent microscopy. Confocal microscopy should be preferred in future studies like this, because it gives higher resolution and better color separation, enables the use of more colors at the same time, and is more accurate in differentiating true colocalization from proteins in close proximity.

Also, being a descriptive morphological study, our study did not deal with the ability of the cells to synthesize and secrete lactose in culture. This is a complex issue that is not dependent only on the presence of lactogenic hormones in the medium, and may be affected by many factors, such as the intracellular matrix. However, if these results were obtained in cells expressing lactose, then the issues of cell-to-cell variations and possible phenotypic effects discussed above would have been minimized.

The fact that this work is based on two previous works, one in vivo on mammary glands from lactating mice16 and the second in vitro on the same CIT3 cells, 25,55 establishes a continuum that supports our results within their limited scope. These results can possibly form the basis and methodological approach for future works in other primary MEC in order to try and generalize the conclusions.

In summary, our results demonstrated a basal constitutive GLUT1 membrane-recycling pathway between an intracellular pool and the cell surface in CIT3 MMEC, which targets most of the GLUT1 to the plasma membrane in GM. As in other cell types it is responsible for maintaining basal glucose uptake. But, in these MEC there is hormonally regulated cell type-specific, developmental stage-specific sorting machinery for GLUT1 intracellular targeting in lactation. This process is induced by prolactin and is highly sensitive to low concentrations of prolactin. It provides the cell with a quick mechanism by which it can supply free glucose intracellularly to serve as substrate for lactose synthesis in the Golgi. This machinery offers another level of regulation of lactose synthesis by altering GLUT1 targeting within minutes to hours, as was demonstrated also in vivo.16 The rapid responsiveness of GLUT1 targeting suggests that this machinery does not require new protein synthesis. It may also support glucose transport as a rate-limiting step for lactose synthesis during lactation.


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The Angelina Jolie Effect in Jewish Law: Prophylactic Mastectomy and Oophorectomy in BRCA Carriers
Sharon Galper Grossman
Rambam Maimonides Med J 2015 October;6(4):e0037

Background: Following the announcement of actress Angelina Jolie’s prophylactic bilateral mastectomies and subsequent prophylactic oophorectomy, there has been a dramatic increase in interest in BRCA testing and prophylactic surgery.

Objective: To review current medical literature on the benefits of prophylactic mastectomy and oophorectomy among BRCA-positive women and its permissibility under Jewish law.
Results: Recent literature suggests that in BRCA-positive women who undergo prophylactic oophorectomy the risk of dying of breast cancer is reduced by 90%, the risk of dying of ovarian cancer is reduced by 95%, and the risk of dying of any cause is reduced by 77%. The risk of breast cancer is further reduced by prophylactic mastectomy. Prophylactic oophorectomy and prophylactic mastectomy pose several challenges within Jewish law that call into question the permissibility of surgery, including mutilation of a healthy organ, termination of fertility, self-wounding, and castration. A growing number of Jewish legal scholars have found grounds to permit prophylactic surgery among BRCA carriers, with some even obligating prophylactic mastectomy and oophorectomy.
Conclusion: Current data suggest a significant reduction in mortality from prophylactic mastectomy and oophorectomy in BRCA carriers. While mutilation of healthy organs is intrinsically forbidden in Jewish law, the ability to preserve human life may contravene and even mandate prophylactic surgery.
INTRODUCTION In May 2013, the widely acclaimed Hollywood celebrity, Angelina Jolie, published an op-ed in The New York Times announcing that her mother, grandmother, and aunt had had cancer, that she had tested positive for the BRCA mutation, and that she had undergone bilateral prophylactic mastectomies to prevent breast cancer.1 This announcement led to what oncologists refer to as “The Angelina Jolie Effect,” a more than doubling in the demand for BRCA testing in women who would not otherwise have gone for testing, but were at high risk for carrying the mutation based on family history and therefore should have undergone genetic testing.2 In March 2015, Angelina Jolie published a second oped in The New York Times disclosing that she had undergone laparoscopic oophorectomy, removal of the ovaries to prevent ovarian cancer, and that she was receiving hormone replacement therapy to prevent the side-effects of premature menopause.3

SCIENTIFIC BACKGROUND Hereditary breast cancer accounts for 5%–10% of all breast cancer.4–7 The vast majority of inherited breast cancers are due to mutations in two breast cancer genes referred to as BRCA1 and 2.6 The risks of developing breast and ovarian cancer are higher in carriers of the BRCA1 mutation compared to carriers of BRCA2.8 In addition, cancer is more likely to occur at a younger age in carriers of BRCA1 mutations than in carriers of BRCA2. An average woman has a 12% lifetime risk of developing breast cancer.9 In a recent population-based study of Ashkenazi Jews in Israel, regardless of family history, the risks of developing breast cancer among BRCA1 and 2 carriers were 60% and 40%, respectively, and the risks of developing ovarian cancer were 53% and 62%, respectively.10 These results are consistent with the findings of a meta-analysis of 10 studies of patients in high-risk clinics which reported that the risks of developing breast cancer by the age of 70 in BRCA1 and 2 carriers are 57% and 49%, respectively, and the risks of ovarian cancer are 40% and 18%, respectively.8 These risks are significantly higher among women born more recently than among women born earlier, a birth cohort effect presumably due to modifications in non-genetic factors such as earlier menarche and later childbearing.10
Possible interventions for BRCA carriers might include increased surveillance, chemoprevention, and prophylactic surgery. Surveillance for breast cancer has consisted of MRI and mammogram beginning at age 25 or individualized to 10 years before the first cancer diagnosed in the family. While the addition of MRI to mammogram increases cancer detection rates and diagnoses cancer at an earlier stage, this strategy has not been shown to prolong survival in BRCA carriers.11–14 Surveillance for ovarian cancer has consisted of trans-vaginal ultrasound and blood tests for elevated tumor markers such as CA-125. However, this strategy has not been found to be effective.15,16 In fact, the National Cancer Institute does not recommend surveillance for ovarian cancer among BRCA carriers.
Another approach to reducing the risk of cancer among BRCA carriers is chemoprevention: tamoxifen to reduce the risk of breast cancer, and oral contraceptive pills to reduce the risks of ovarian cancer. Tamoxifen appears to be effective in reducing breast cancer in carriers of BRCA2 but not in carriers of BRCA1.17,18 The differential effect of tamoxifen may be due to the differential expression of the estrogen receptor in tumors of BRCA carriers. The BRCA1 carriers tend to develop estrogen receptor-negative breast cancers which do not depend on estrogen to grow, and the BRCA2 carriers tend to develop breast cancers that are estrogen receptor-positive and depend on estrogen to grow.19–21 Chemoprevention with tamoxifen has not been shown to prolong survival in BRCA2 carriers.17,18 Oral contraceptive pills have been shown to reduce the chances of developing ovarian cancer in BRCA carriers by 50% without increasing the risk of breast cancer, but this intervention has not been shown to reduce the chances of dying of ovarian cancer.22
PROPHYLACTIC SURGERY Prophylactic surgery consists of mastectomy, removal of the breasts to prevent breast cancer, and oophorectomy, removal of the ovaries to prevent ovarian cancer. Prophylactic mastectomy reduces the chances of developing breast cancer by 90% or more.23–30 In one series, there were no cases of breast cancer 3 years after prophylactic surgery.29 Prophylactic oophorectomy reduces the chances of developing ovarian cancer by 80% and can also reduce the chances of developing breast cancer by 50%.31–35 Carriers of the BRCA mutation may opt for mastectomy alone with surveillance of the ovaries (as Angelina Jolie chose to do between 2013 and 2015), prophylactic oophorectomy alone which will reduce the chances of developing both breast and ovarian cancer, or prophylactic mastectomy and oophorectomy (which Angelina Jolie ultimately chose to do). The ideal age to perform prophylactic oophorectomy is not known, but the recommendation is to complete childbearing by age 35–40 and then undergo prophylactic surgery as there is concern that delaying oophorectomy would increase the chances of developing ovarian cancer.35 In addition, the magnitude of the protective effects of oophorectomy in reducing the chances of breast cancer is greater the younger the age of oophorectomy.29,36 Oophorectomy at this age does cause premature menopause; however, as Angelina Jolie has illustrated, these symptoms can safely be managed with short-term hormone replacement therapy.37–39
How do BRCA carriers cope with prophylactic surgery? Emerging data would suggest that overall quality of life for BRCA carriers who undergo prophylactic surgery is not compromised by surgery. The BRCA carriers who undergo prophylactic surgery report high satisfaction with surgery and less cancer worry than BRCA carriers who opt for surveillance.40,41 However, prophylactic surgery may be associated with sexual dysfunction and menopausal symptoms which can be addressed by proper medical intervention. Although it was initially thought, based on theoretical modeling, that BRCA carriers who underwent prophylactic surgery would live longer than those who opted for surveillance, this was not based on actual prospective patient data.42 Recently, a number of studies have confirmed that BRCA carriers who undergo prophylactic surgery live longer than those who undergo surveillance.29,35,43 In one series, the chances of dying of ovarian cancer were reduced by 95% among BRCA carriers who opted for prophylactic oophorectomy compared to carriers who opted for surveillance, the chances of dying of breast cancer were reduced by 90%, and the chances of dying of any cause were reduced by 70%.29 Previously, it was assumed that prophylactic oophorectomy would reduce the chances of a BRCA carrier dying of ovarian cancer but that inducing premature menopause would be harmful and negate any beneficial effects of preventing ovarian cancer. Physicians believed that the BRCA carrier who opted for prophylactic oophorectomy was “trading” ovarian cancer for a host of new medical problems associated with entering premature menopause for no “net” benefit. Yet, the most recent studies show that, overall, BRCA carriers who undergo prophylactic surgery live longer than those who opt for surveillance, demonstrating that, regardless of what medical problems premature menopause may cause, the net effect of prophylactic surgery is that carriers who undergo such surgery live longer. The medical benefits clearly outweigh the risks.
HALACHIC ISSUES From a Halachic perspective, there are several concerns arguing against prophylactic surgery (Table 1). First of all, prophylactic surgery involves mutilation of a healthy organ. Secondly, removing a healthy organ—especially one that defines a woman’s sexuality and her appearance—may cause significant psychological distress, although the available quality of life data would suggest that from a psychological perspective this surgery is well tolerated.40,41
In addition, there are two other potential Halachic concerns regarding removal of ovaries. First, removal of ovaries may prevent the BRCA carrier from fulfilling the mitzvah to “be fruitful and multiply” (pru u’revu). Second, removal of ovaries may violate the prohibition against castration (sirus). Arguing in favor of prophylactic surgery are new, emerging, compelling medical data showing that BRCA carriers who undergo prophylactic surgery are less likely to die of cancer and more likely to live longer than women who opt for surveillance. Angelina Jolie’s decision prophylactically to remove her healthy breasts and most recently her healthy ovaries raises several Halachic questions including the following: (1) is there a Halachic obligation to prevent disease; (2) is it permitted for a BRCA carrier to remove a healthy organ; (3) does prophylactic surgery violate the prohibition against harming one’s body (chovel); (4) does prophylactic oophorectomy prevent the BRCA carrier from fulfilling the mitzvah to procreate; (5) does prophylactic oophorectomy violate the prohibition against castration; (6) is it permitted to perform prophylactic surgery in a BRCA carrier; (7) is a BRCA carrier obligated by Halacha to undergo prophylactic surgery; and, lastly, (8) are we as a Jewish society, particularly in Israel, obligated to pay for prophylactic surgery in BRCA carriers?
In summary, there is a clear Halachic obligation to prevent disease. It is permitted to remove a healthy body part to prevent disease in the future. Prophylactic oophorectomy interferes with obligations to procreate and prohibitions of castration. Oophorectomy after completing childbearing helps eliminate issues relating to “procreation.” When performed after menopause, prophylactic oophorectomy may obviate the prohibition against castration. Ultimately, saving a life overrides castration and any prohibitions including the prohibition against harming one’s body. Given the emerging data favoring prophylactic surgery, a growing number of Jewish arbiters believe that prophylactic surgery is Halachically permitted, with some positing that a BRCA carrier is obligated to undergo prophylactic surgery.
Angelina Jolie’s Status in Judaism Angelina Jolie’s very public medical journey has increased awareness of the BRCA mutation and the demand for testing in high-risk women who would not otherwise have been tested. In addition, she has increased interest in potentially life-saving prophylactic surgery. It is not possible to measure how many lives she has saved by making her very personal, medical odyssey public. Regardless of her other behaviors and politics, her decision to publicize her status as a BRCA carrier and her decisions to undergo prophylactic surgery make her worthy of the description in Sanhedrin, “Whoever saves one Jewish life is considered to have created an entire world.”63
Angelina Jolie has created many worlds, and for this we as a Jewish people must be eternally grateful.

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Lonely Receptors: RXR – Jensen, Chambon, and Evans

Larry H. Bernstein, MD, FCAP, Curator

Leaders in Pharmaceutical Intelligence

Series E. 2; 7.2


Nuclear receptors provoke RNA production in response to steroid hormones

Albert Lasker Basic Medical Research Award

Pierre Chambon, Ronald Evans and Elwood Jensen

For the discovery of the superfamily of nuclear hormone receptors and elucidation of a unifying mechanism that regulates embryonic development and diverse metabolic pathways.

Hormones control a vast array of biological processes, including embryonic development, growth rate, and body weight. Scientists had known since the early 1900s that tiny hormone doses dramatically alter physiology, but they had no idea that these signaling molecules did so by prodding genes. The 1950s, when Jensen began his work, was the great era of enzymology. Conventional wisdom held that estradiol—the female sex hormone that instigates growth of immature reproductive tissue such as the uterus—entered the cell and underwent a series of chemical reactions that produced a particular compound as a byproduct. This compound—NADPH—is essential for many enzymes’ operations but its small quantities normally limit their productivity. A spike in NADPH concentrations would stimulate growth or other activities by unleashing the enzymes, the reasoning went.

In 1956, Jensen (at the University of Chicago) decided to scrutinize what happened to estradiol within its target tissues, but he had a problem: The hormone is physiologically active in minute quantities, so he needed an extremely sensitive way to track it. He devised an apparatus that tagged it with tritium—a radioactive form of hydrogen—at an efficiency level that had not previously been achieved. This innovation allowed him to detect a trillionth of a gram of estradiol.

When he injected this radioactive substance into immature rats, he noticed that most tissues—skeletal muscle, kidneys and liver, for example—started expelling it within 15 minutes. In contrast, tissues known to respond to the hormone—those of the reproductive tract—held onto it tightly. Furthermore, the hormone showed up in the nuclei of cells, where genes reside. Something there was apparently grabbing the estradiol.

Jensen subsequently showed that his radioactive hormone remained chemically unchanged once inside the cell. Estrogen did not act by being metabolized and producing NADPH, but presumably by performing some job in the nucleus. Subsequent work by Jensen and Jack Gorski established that estradiol converts a protein in the cytoplasm, its receptor, into a form that can migrate to the nucleus, embrace DNA, and turn on specific genes.

From 1962 to 1980, molecular endocrinologists built on Jensen’s work to discover the receptors for the other major steroid hormones—testosterone, progesterone, glucocorticoids, aldosterone, and the steroid-like vitamin D. In addition to Jensen and Gorski, many scientists—notably Bert O’Malley, Jan-Ake Gustafsson, Keith Yamamoto, and the late Gordon Tompkins—made crucial observations during the early days of steroid receptor research.

Clinical Applications of Estrogen-Receptor Detection

Clinicians knew that removing the ovaries or adrenal glands of women with breast cancer would stop tumor growth in one out of three patients, but the molecular basis for this phenomenon was mysterious. Jensen showed that breast cancers with low estrogen-receptor content do not respond to surgical treatment. Receptor status could therefore indicate who would benefit from the procedure and who should skip an unnecessary operation. In the mid-1970s, Jensen and his colleague Craig Jordan found that women with cancers that contain large amounts of estrogen receptor are also likely to benefit from tamoxifen, an anti-estrogen compound that mimics the effect of removing the ovaries or adrenal glands. The other patients—those with small numbers of receptors—could immediately move on to chemotherapy that might combat their disease rather than waiting months to find out that the tumors were growing despite tamoxifen treatment. By 1980, Jensen’s test had become a standard part of care for breast cancer patients.

In the meantime, Jensen set about generating antibodies that bound the receptor—a tool that provided a more reliable way to measure receptor quantities in excised breast tumor specimens. His work has transformed the treatment of breast cancer patients and saves or prolongs more than 100,000 lives annually.

Long-Lost Relatives

By the early 1980s, interest in molecular endocrinology had shifted toward the rapidly developing area of gene control. Chambon and Evans had long wondered how genes turn on and off, and recognized nuclear hormone signaling as the best system for studying regulated gene transcription. They wanted to know exactly how nuclear receptors provoke RNA production in response to steroid hormones. To manipulate and analyze the receptors, they would need to isolate the genes for them.

By late 1985 and early 1986, Evans (at the Salk Institute in La Jolla) and Chambon (at the Institute of Genetics and Molecular and Cellular Biology in Strasbourg, France) had pieced together the glucocorticoid and estrogen receptor genes, respectively. They noticed that the sequences resembled that of v-erbA, a miscreant viral protein that fosters uncontrolled cell growth. This observation raised the possibility that v-erbA and its well-behaved cellular counterpart, c-erbA, would also bind DNA and control gene activity in response to some chemical activator, or ligand. In 1986, Evans and Björn Vennström simultaneously reported that c-erbA was a thyroid hormone receptor that was related to the steroid hormone receptors, thus uniting the fields of thyroid and steroid biology.

Chambon and Evans set to work deconstructing the glucocorticoid and estrogen receptors. By creating mutations at different spots and probing which activities the resulting proteins lost, they dissected the receptor into three domains: one bound hormone, one bound DNA, and one activated target genes. The structure of each domain strongly resembled the analogous one in the other receptor.

Chambon and Evans wanted to match other members of the growing receptor gene family with their chemical triggers. Because the DNA- and ligand-binding regions functioned independently, it was possible to hook the DNA-binding domain of, say, the glucocorticoid receptor to the ligand-binding domain of another receptor whose ligand was unknown. The ligand for that receptor would then activate a glucocorticoid-responsive test gene.

Evans would use this method to identify ligands for several novel members of the nuclear receptor family, and both he and Chambon exploited it to discover a physiologically crucial receptor. In the late 1970s, scientists had suggested that the physiologically active derivative of vitamin A, retinoic acid, could exert its effects by binding to a nuclear receptor. This nutrient is essential from fertilization through adulthood, and researchers were eager to understand its activities on a molecular level. During embryonic development, deficiency of retinoic acid impairs formation of most organs, and the compound can hinder cancer cell proliferation. So Chambon set out to find a receptor that responded to retinoic acid. He isolated a member of the nuclear receptor gene family whose production increased in breast cancer cells that slowed their growth upon exposure to the chemical. Simultaneously, Evans identified the same protein. He tested whether more than a dozen compounds activated an unknown receptor and one passed: retinoic acid.

Remarkably, in 1986, the two scientists had independently—and unbeknownst to each other—identified the same retinoic acid receptor, a molecule of tremendous significance. The discovery of this molecule provided an entry point for detailing vitamin A biology.

Rx for Lonely Receptors: RXR

The list of presumptive nuclear receptors was growing quickly as scientists realized that the common DNA sequences provided a handle with which to grab these molecules from the genome. Because their chemical activators weren’t known, they were called “orphan” receptors, and researchers were keen on “adopting” them to ligands. Some of these ligands, they reasoned, would represent previously unknown classes of gene activators. The test system that Chambon and Evans used to match up retinoic acid with its receptor, in which they stitched an unknown ligand-binding domain to a DNA-binding domain for a receptor with known target sequences, could be harnessed to accomplish this task.

Evans had identified some potential nuclear receptors from fruit flies. He decided to pursue a human orphan receptor that closely resembled one of these receptor genes, reasoning that a protein that functioned in both flies and mammals was likely to perform an important job.

This receptor responded to retinoic acid in intact cells but did not bind it in the test tube, so Evans called it the Retinoid X Receptor (RXR), thinking that its ligand was some retinoic acid derivative. In cells, enzymes convert retinoic acid to metabolites and it seemed possible that one of these compounds was RXR’s ligand. In 1992, Evans’s group and one at Hoffmann-La Roche discovered that 9-cis-retinoic acid, a stereoisomer of retinoic acid, could activate RXR, identifying the first new receptor ligand in 25 years. This finding launched the orphan receptor field because it provided strong evidence that the strategy could unearth previously unknown ligands.

In the meantime, Chambon had found that the purified retinoic acid receptor, in contrast to the estrogen receptor, did not bind efficiently to its target DNA. Other nuclear receptors, too, needed help grasping genes. In the test tube, the retinoic acid, thyroid hormone, and vitamin D3 receptors could attach well to their target DNA only when supplemented with cellular material, which presumably contained some crucial substance. Chambon and Michael Rosenfeld independently purified a single protein that performed this feat, and it turned out to be none other than RXR. This ability of RXR to pair with other receptors—forming so-called heterodimers—would turn out to be key for switching on many orphan receptors. These heterodimeric couplings yield large numbers of distinct gene-controlling entities.

Chambon revealed the power of mixing and matching in these molecular duos through his thorough and extensive genetic manipulations in mice. He has shown that vitamin A exerts its wide-ranging effects on organ development in the embryo through the action of eight different forms of the retinoic acid receptor and six different forms of RXR, interacting with each other in a multitude of combinations.

Clinical Applications of the Superfamily Work

The concept of RXR as a promiscuous heterodimeric partner for certain nuclear receptors led to the unexpected identification of a number of clinically relevant receptors. These proteins include the peroxisome proliferator-activated receptor (PPAR), which stimulates fat-cell maturation and sits at the center of Type 2 diabetes and a number of lipid-related disorders; the liver X receptors (LXRs) and bile acid receptor (FXR), which help manage cholesterol homeostasis; and the steroid and xenobiotic receptor (PXR), which turns on enzymes that dispose of chemicals that need to be detoxified, such as drugs.

Because the nuclear receptors wield such physiological power, they have provided excellent targets for disease treatment. The anti-diabetes compounds glitazones, for example, work by stimulating PPAR, and the clinically used lipid-lowering medications called fibrates work by binding a closely related receptor, PPAR. Retinoic acid therapy has dramatically altered the prognosis of people with acute promyelocytic leukemia by triggering specialization of the immature white blood cells that accumulate in these individuals. The three-dimensional structure of nuclear receptors with and without their ligands, which Chambon and his colleagues first solved, promises to accelerate drug discovery in the whole field.

Nuclear hormone receptors have touched on human health in other ways as well. Genetic perturbations in the genes for these proteins cause a variety of illnesses. For example, certain forms of rickets arise from mutations in the vitamin D receptor and several disorders of male sexual differentiation stem from defects in the androgen receptor.

The discoveries of Jensen, Chambon, and Evans revealed an unimagined superfamily of proteins. At the start of this work almost 50 years ago, no one would have anticipated that steroids, thyroid hormone, retinoids, vitamin D, fatty acids, bile acids, and many lipid-based drugs transmit their signal through similar pathways. Four dozen human nuclear receptors are now known, and scientists are working out the roles of these proteins in normal and aberrant physiology. These discoveries have revolutionized the fields of endocrinology and metabolism, and pointed toward new tactics for drug discovery.

by Evelyn Strauss, Ph.D.


The 2004 Lasker Award for Basic Medical Research will be presented to Elwood Jensen, Ph.D., the Charles B. Huggins Distinguished Service Professor Emeritus in the Ben May Institute for Cancer Research at the University of Chicago, one of three scientists whose discoveries “revolutionized the fields of endocrinology and metabolism,” according to the award citation. Jensen’s work had a rapid, direct and lasting impact on treatment and prevention of breast cancer.

The Lasker Awards are the nation’s most distinguished honor for outstanding contributions to basic and clinical medical research. Often called “America’s Nobels,” the Lasker Award has been awarded to 68 scientists who subsequently went on to receive the Nobel Prize, including 15 in the last 10 years.

Jensen will share the basic medical research award with two colleagues, Pierre Chambon, of the Institute of Genetics and Molecular and Cellular Biology (Strasbourg, France), and Ronald M. Evans of the Salk Institute for Biological Studies (La Jolla, California) and the Howard Hughes Medical Institute.

They were selected for their discovery of the “superfamily of nuclear hormone receptors and the elucidation of a unifying mechanism that regulates embryonic development and diverse metabolic pathways.” The implications of this research for understanding human disease and accelerating drug discovery “have been profound and hold much promise for the future,” notes the announcement from the Lasker Foundation.

Jensen is being honored for his pioneering research on how steroid hormones, such as estrogen, exert their influence. His discoveries explained how these hormones work, which has led to the development of drugs that can enhance or inhibit the process.

Hormones control a vast array of biological processes, including embryonic development, growth rate and body weight. Before Jensen, however, the way which hormones cause these effects was “a complete mystery,” recalled Gene DeSombre, Ph.D., professor emeritus at the University of Chicago, who worked with Jensen in the Ben May Institute as a post-doctoral fellow and then as a colleague.

In the 1950s, biochemists thought a hormone entered a cell, where a series of oxidation and reductions reactions with the estrogen provided needed energy for the growth stimulation and other specific actions shown by estrogens.

From the late 1950s to the 1970s Jensen entirely overturned that notion. Working with estrogen, he proved that hormones do not undergo chemical change. Instead, they bind to a receptor protein within the cell. This hormone-receptor complex then travels to the cell nucleus, where it regulates gene expression.

At the time, this idea was heresy. “That really got him into some hot water,” recalled DeSombre. “Jensen struggled quite a lot,” echoes Shutsung Liao, Ph.D., another Ben May colleague, who subsequently found a similar system for testosterone action. But for Jensen, just getting into hot water was a struggle. When he first presented preliminary data at a 1958 meeting in Vienna, only five people attended, three of whom were the other speakers. More than 1,000 attended a simultaneous symposium on the metabolic processing of estrogen.

In the next 20 years, Jensen convinced his colleagues by publishing a series of major and highly original discoveries in four related areas of hormone research:

  • Jensen discovered the estrogen receptor, the first receptor found for any hormone. In 1958, using a radioactive marker, he showed that only the tissues that respond to estrogen, such as those of the female reproductive tract, were able to concentrate injected estrogen from the blood. This specific uptake suggested that these cells must contain binding proteins, which he called “estrogen receptors.”
  • In 1967, Jensen and Jack Gorski of the University of Wisconsin showed that these putative receptors were macromolecules that could be extracted from these tissues. With this method, Jensen showed that when estrogen bound to this receptor, the compound then migrated to the nucleus where it bound avidly and activated specific genes, stimulating new RNA synthesis.
  • By 1968, Jensen had devised a reliable test for the presence of estrogen receptors in breast cancer cells. It had been known for decades that about one-third of premenopausal women who had advanced breast cancer would respond to estrogen blockade brought about by removing their ovaries, the source of estrogen, but there was no way to predict which women would respond. In 1971, Jensen showed that women with receptor-rich breast cancers often have remissions following removal of the sources of estrogen, but cancers that contain few or no estrogen receptors do not respond to estrogen-blocking therapy.
  • By 1977, Jensen and Geoffrey Greene, Ph.D., also in the University of Chicago’s Ben May Institute, had developed monoclonal antibodies directed against estrogen receptors, which enabled then to quickly and accurately detect and count estrogen receptors in breast and other tumors. By 1980, this test had become a standard part of care for breast cancer patients

This work “transformed the treatment of breast cancer patients,” notes the Lasker Foundation, “and saves or prolongs more than a 100,000 lives annually.”

”Jensen’s revolutionary discovery of estrogen receptors is beyond doubt one of the major achievements in biochemical endocrinology of our time,” said DeSombre. “His work is hallmarked by great technical ingenuity and conceptual novelty. His promulgation of simple yet profound ideas concerning the role of receptors in estrogen action have been of the greatest importance for research on the basic and clinical physiology not only of estrogens but also of all other categories of steroid hormones.”

By the early 1970s, Jensen was searching for chemical, rather than surgical, ways to shield estrogen-dependent tumors from circulating hormones. He and colleague Craig Jordan (then at the Worcester Foundation for Experimental Biology in Massachusetts) subsequently found that women with cancers that contain large amounts of estrogen receptor are also likely to benefit from tamoxifen, a compound that blocks some of the effects of estrogen. Patients with few or no receptors could immediately move on to chemotherapy rather than waiting months to find out that the tumors were growing despite tamoxifen treatment.

Following Jensen’s lead, researchers soon found that the receptors for the other major steroid hormones, such as testosterone, progesterone, and cortisone, worked essentially the same way.

In 1986, Pierre Chambon and Ronald Evans separately but simultaneously discovered that the steroid hormone receptors were merely the tip of the iceberg of what would turn out to be a large family of structurally related nuclear receptors, now known to consist of 48 members. Evans and Chambon unearthed a number of these receptors, which revealed new regulatory systems that control the body’s response to essential nutrients (such as Vitamin A), fat-soluble signaling molecules (such as fatty acids and bile acids), and drugs (such as the glitazones used to treat Type 2 diabetes and retinoic acid for certain forms of acute leukemia).

These three individuals “created the field of nuclear hormone receptor research, which now occupies a large area of biological and medical investigation,” said Dr. Joseph L. Goldstein, chairman of the international jury of researchers that selects recipients of the Lasker Awards, and recipient of the Lasker Award for Basic Medical Research and the Nobel Prize in Medicine in 1985.

They revealed the “unexpected and unifying mechanism by which many signaling molecules regulate a plethora of key physiological pathways that operate from embryonic development through adulthood. They discovered a family of proteins that allows chemicals as diverse as steroid hormones, Vitamin A, and thyroid hormone to perform in the body.”

Jensen, known for concluding his lectures in verse, neatly summed up what his extraordinary series of discoveries might mean to a woman who has been diagnosed with breast cancer:

“A lady with growth neoplastic
Thought surgical ablation too drastic.
She preferred that her ill
Could be cured with a pill,
Which today is no longer fantastic.”


Nuclear Receptors in Biology and Diseases

Thematic Minireview Series on Nuclear Receptors in Biology and Diseases

Sohaib Khan and Jerry B Lingrel

Although a connection between breast cancer and the ovary was made by Sir George Beatson in 1896 and estrogen was purified in 1920, it remained puzzling as to how the hormone exerted its biological effects. In the late 1950s, when Elwood Jensen delved into this problem by asking, essentially, “What does tissue do with this hormone?” little did he know that his quest would lead to the establishment of the nuclear receptor field. The late 1950s was the era of intermediary metabolism and enzymology, when steroid hormones were considered likely substrates in the formation of metabolites that functioned as cofactors in an essential metabolic pathway. The biological responses to estrogens and other steroids were thought to be mediated by enzymes. Against this background and prevailing dogma, Jensen and colleagues defined the biochemical mechanisms by which steroid hormones exert their effects. While working at the University of Chicago’s Ben May Institute for Cancer Research, they synthesized tritium-labeled estradiol and concurrently developed a new method to measure its uptake in biological material. These tools enabled them to determine the biochemical fate of physiological amounts of hormone. They discovered that the reproductive tissues of the immature rat contain characteristic hormone-binding components with which estradiol reacts to induce uterine growth without itself being chemically changed. From the close correlation between the inhibition of binding and inhibition of growth response, Jensen established that the binding substances were receptors. Thus, we saw the birth of the first member of the nuclear receptor family (known as the estrogen receptor). These findings stimulated the search for other physiological receptors, and the pioneering works by Pierre Chambon, Ronald Evans, Jan-Åke Gustafsson, Bert W. O’Malley, and Keith Yamamoto led to the discoveries of the glucocorticoid receptor (GR),2 progesterone receptor, retinoic acid receptor, and orphan receptors. In a rather short span of time, the nuclear receptor family has grown into a 49-member-strong “superfamily.” This is a family whose members, functioning as sequence-specific transcription factors, have defined the many intricacies of the mechanism of transcription. These ligand-dependent transcription factors generally possess similar “domain organizations,” of which the DNA-binding domain and the ligand-binding domain are critical in amplifying the hormonal signals via the receptor target genes. The nuclear receptor family is divided into four groups: (i) Group 1 is composed of steroid hormone receptors that control target gene transcription by binding as homodimers to response element (RE) palindromes; (ii) in Group 2, the nuclear receptors heterodimerize with retinoid X receptor and generally bind to direct repeat REs; (iii) Group 3 consists of those orphan receptors that function as homodimers and bind to direct repeat REs; and (iv) orphan receptors in Group 4 function as monomers and bind to single REs.

Since the early demonstration by Jack Gorski and Jensen that the estrogen receptor (ER) activates transcription, the nuclear receptor field has come a long way. In addition to the first cloning of the polymerase II transcription factors (GR and ER cDNAs), of note is the discovery of steroid receptor coactivators (SRCs), a truly major piece of the transcriptional jigsaw puzzle, described by the laboratories of O’Malley and Myles Brown. The induction of coactivators and corepressors in the transcriptional machinery has expanded tremendously our understanding of this complex process. We now know that ligand binding to the respective receptors triggers a fascinating chain of events, including the translocation of the receptors to the nucleus, ligand-induced changes in the receptor conformations, receptor dimerization, interaction with the target gene promoter elements, recruitment of coactivators (or corepressors), chromatin remodeling, and subsequent interaction with the polymerase II complex to initiate transcription.

By virtue of their abilities to regulate a myriad of human developmental and physiological functions (reproduction, development, metabolism), nuclear receptors have been implicated in a wide range of diseases, such as cancer, diabetes, obesity, etc. Not surprisingly, drug companies are spending billions of dollars to develop medicines for cancer and metabolic disorders that involve nuclear receptors. More than 50 years after the discovery of the ER, the scientific community owes Jensen and other founding members of the nuclear receptor family much gratitude, for they have taken us through a remarkable expedition filled with eureka moments to understand how hormones and other ligands function!

This thematic minireview series will cover a range of topics in the nuclear receptor field. The minireviews include the current studies of identifying subtypes of the GR. Different receptors arise from alternative mRNA splicing and from the use of different promoter start sites and post-translational modifications, such as phosphorylation. The series covers the physiological roles of the different GRs. The field of orphan nuclear receptors and the search for possible ligands also are reviewed. One minireview concentrates largely on the following nuclear receptors: peroxisome proliferator-activated receptor (PPAR) α, PPARγ, Rev-erbα, and retinoic acid receptor-related orphan receptor α. ERα was the first identified and has been studied the most, whereas ERβ has not been studied in the same detail. ERβ is very important, and one of the minireviews provides a summary of the new biological functions that are being ascribed to it. Also, the development of small molecule inhibitors for the ER will be considered. An important aspect of nuclear receptor function is how these receptors function in transcription. The role of transcriptional coactivators in nuclear receptor gene regulation will be reviewed as well as how signal amplification and interaction are involved in transcription regulation by steroids. The SRC/p160 family of coregulators includes SRC-1, SRC-2, and SRC-3, and the latter has been shown to act as an oncogene, particularly in breast cancer. Molecular analysis of its role in breast cancer progression and metastasis will be the focus of one of the minireviews. In addition, interactions of nuclear receptors with the genome will be reviewed, as will the role of the homeodomain protein HoxB13 in specifying the cellular response to androgens. Mining nuclear receptor cistromes and how nuclear receptors reset metabolism also will be considered. The association of nuclear receptors (e.g. PPARδ) with physiological functions, such as circadian rhythm and muscle functions, will also be addressed. Finally, the role of nuclear receptors in disease using the retinoid X receptor α/β knock-out and transgenic mouse model skin syndromes and asthma will be reviewed. These are diverse and important topics that are critical in understanding the regulation of nuclear receptors and the biological roles they play in normal function and disease.

The Nuclear Receptor Superfamily: A Rosetta Stone for Physiology

Ronald M. Evans
Howard Hughes Medical Institute, Gene Expression Laboratory, The Salk Institute for Biological Studies, La Jolla, California 92037
Molecular Endocrinology 19(6):1429–143

In the December 1985 issue of Nature, we described the cloning of the first nuclear receptor cDNA encoding the human glucocorticoid receptor (GR) (1). In the 20 yr since that event, our field has witnessed a quantum leap by the subsequent discovery and functional elaboration of the nuclear receptor superfamily (2)—a family whose history is linked to the evolution of the entire animal kingdom and whose actions, by decoding the genome, span the vast diversity of biological functions from development to physiology, pathology, and treatment. A messenger is an envoy or courier charged with transmitting a communication or message. In one sense, the cloning of that first messenger (the GR) represented the completion of a prediction that began with Elwood Jensen’s characterization of the first steroid receptor protein (3) and continued with the pioneering work of others in the steroid receptor field (including Gorski, O’Malley, Gustafsson, and Yamamoto). Yet, like the discovery of the Rosetta stone in 1799, the revelation of the GR sequence heralded a completely unpredictable demarcation in the field, helping to solve mysteries unearthed nearly 100 yr ago as well as opening a portal to the future. The beginnings of the adventure lie in disciplines such as medicine and nutrition, which gave rise to the emergent field of endocrinology in the first half of the last century. The purification of chemical messengers ultimately known as hormones from organs and vitamins from foods spurred the study of these compounds and their physiologic effects on the body. At about the same time, the field of molecular biology was emerging from the disciplines of chemistry, physics, and their application to biological problems such as the structure of DNA and the molecular events surrounding its replication and transcription. It would not be until the late 1960s and 1970s that endocrinology and molecular biology would begin to intersect as the link between receptors and transcriptional control were being laid down. During this time, the work of Jensen (4) and Gorski (5) identified a high-affinity estrogen receptor (ER) that suggested an action in the nucleus. Gordon Tomkins and his associates (J. Baxter, G. Ringold, E. B. Thompson, H. Samuels, H. Bourne, and others) were one of the most creative forces studying glucocorticoid action (6). Concurrent work by O’Malley, Gustafsson, and Yamamoto provided further, important evidence supporting a link between steroid receptor action and transcription (see accompanying perspective articles in this issue of Molecular Endocrinology). But whereas the steroid hormone field continued to evolve in this direction, it is of interest to note that the mechanism of action of thyroid hormone and retinoids remained clouded and controversial until the eventual cloning of their receptors in the late 1980s. Likewise, no one had foreseen the possibility that other lipophilic molecules (like oxysterols, bile acids, and fatty acids) would also function through a similar mechanism, or that other steroid receptor-like proteins existed that would play an important role in transcriptional regulation of so many diverse pathways. Thus, the GR isolation in 1985 led to the concept of a hidden superfamily of receptors that in a very real way provided the needed molecular code to unravel the puzzle of physiologic homeostasis.

Unconventional Gene-Ology

The study of RNA tumor viruses was ascendant, and the concept that they evolved by pirating key signaling pathways greatly influenced my future studies. With this training, I went on to work with Jim Darnell at the Rockefeller University on adenovirus transcription, a model brought to the lab by Lennart Philipson. At the time, adenovirus was one of the best tools to study programmed gene expression in an animal cell. My sole focus was to localize the elusive major late promoter, which provided my first Nature paper (7). Ed Ziff, a newly hired assistant professor from Cambridge, brought innovative unpublished DNA and RNA sequencing techniques that, after much technical angst, allowed us to sequence the major late promoter and derive the structure of the first eukaryotic polymerase II promoter (8). This thrilling result convinced me that the problem of gene control could be solved at the molecular level. Our next goal, which I shared with Michael Harpold in the Darnell lab, was to translate the concepts developed around adenovirus into cellular systems. My model was to analyze the glucocorticoid and thyroid hormone regulation of the GH gene. Under the strict federal guidelines for newly approved recombinant DNA research, we cloned the GH cDNA in 1977 and the first genomic clones in 1978 (9) after I moved on to The Salk Institute. However, to fully address the hormone signaling problem, I realized that it would be necessary to clone the GR and thyroid hormone receptors (TRs), which began in earnest in 1981. Up until that time, the purification and cloning of any polymerase II transcription factor had eluded researchers because of their low abundance. Four years later, the GR would be the first transcription factor for a defined response element to be cloned, sequenced, and functionally identified.

A Rock and A Hard Place

A key question was whether the GR protein encoded by the receptor was sufficient, when expressed in a heterologous cell, to convey the hormonal message. Before the publication, a new postdoc, Vincent Giguere, began tinkering with the isolated GR, trying to address this question. The rate of development of any field is limited by the existing techniques and depends on the development of new ones. Vincent devised a revolutionary technique—the cotransfection assay that required two plasmids to be taken up in the same cell, the expression vector to be transcribed, the encoded protein to be functional and an inducible promoter linked to a chloramphenicol acetyltransferase reporter in the nucleus ready to flicker on (10, 12). With so many variables and unknowns, I was stunned and expressionless when it worked the very first time. Cotransfection was an easy, fast, and quantitative technique. It would become (and still remains) the dominant assay to characterize receptor function. It would also become the mainstay for drug discovery in the pharmaceutical industry. The development of this technique proved a great advantage because existing technology involved creating stable cell lines, a tedious process prone to integration artifacts that ultimately could not match the explosive pace of the field. Indeed, within 4 months Stan and Vincent had fully characterized 27 insertional mutants delineating the DBD, LBD, and two activation domains (12). The route to understanding the signaling mechanism now had a solid structural foundation. A serendipitous gift to my retroviral origins was the homology of the GR sequence to the v-erbA oncogene product of the avian erythroblastosis virus genome (13). With this discovery, erbA advanced to a candidate nuclear transcription factor potentially involved in a signal transduction pathway. Thus, while Stan concentrated on the GR, Cary began to delve into the erbA discovery. Within months of the GR publication, the human c-erbA gene was in hand (14). Unbeknownst to us, Bjorn Vennstrom, one of the first to characterize the avian erythroblastosis virus genome, had also isolated c-erbA and was searching for a function. Based on the low homology of the LBD region to the GR and ER, both groups deduced that the imaginary erbA ligand would be nonsteroidal.

The work of our two groups (15, 16), published in December of 1986, broadened the principles of the signal transduction pathway by demonstrating that thyroid and steroid hormone receptor signaling had a common evolutionary origin and provided an entree to understand how mutations within a receptor could activate it to an oncogene. Although we did not know it at the time, this work would also lead us to the concept of the corepressor. In the meantime, my student, Catherine Thompson, zeroed in on an erb-A-related gene and soon identified a second TR expressed at high levels in the central nervous system (17). Thus came into existence the and forms of the TR. Jeff Arriza, the third graduate student in the lab, purified a genomic fragment that had weakly hybridized to the GR resulting in the isolation of the human mineralocorticoid receptor (MR) (18). MR proved to have an at least 10-fold higher affinity for glucocorticoids than the GR itself and was further distinguished by its ability to bind and be activated by aldosterone. This enabled the development of GR- and MR-selective drugs such as the recent MR antagonist eplerenone. Thus, in a 2-yr time span our lab had progressed on three distinct, albeit related, receptor systems, and in doing so molecular biology and endocrinology were irrevocably linked. The field of molecular endocrinology (and coincidentally the eponymous journal) was born.

Ligands From Stone

I have often been asked how we could handle so many divergent systems. Indeed, from a medical perspective, these systems seem widely unrelated. Studies of ER, progesterone receptor, and androgen receptor (AR) fall under reproductive physiology, vitamin D under bone and mineral metabolism, with vitamin A part of nutritional science. Medical fields are naturally idiosyncratic because of the specialized knowledge required to conduct experiments. With my training as a molecular biologist, physiology was the complex output of genes and thus control of gene expression was the overriding problem. This conceptual approach had a great unifying effect because all receptors transduce their signaling through the gene. As an “outsider,” my goal was to exploit multiple receptor systems to seek general principles. This philosophical approach afforded us a freedom to redefine the signaling problem from the nucleus outward and thus even poorly characterized, even unknown, physiologic systems fell into the crosshairs of our molecular gun.

Vincent, while screening a testes Fig. 1. Models of Nuclear Receptor Structure Top, Original hand-shaped wire model (circa 1992) of the nuclear receptor DBD. Bottom, Schematic representation of the GR DBD. Conserved residues in zinc fingers, P-box and D-box are indicated isolated what would become the vitamin A or retinoic acid receptor (RAR) (19). Initially, Vincent thought he had isolated the AR, although this later proved not to be the case. By that stage, the lab had perfected a new technique—the domain swap—by which the GR DBD could be introduced into any receptor and confers on the chimeric protein the ability to activate a mouse mammary tumor virus reporter. This clever technique, independently developed in the Chambon lab, would prove to be essential. Effectively, the domain swap would enable us to screen for ligands without any knowledge of their physiologic function. Activation of a target gene was all that was needed! By creating this GR chimera, Vincent was able to screen the new receptor against a ligand cocktail including androgens, steroids, thyroid hormone, cholesterol, and the vitamin A metabolite retinoic acid. From the first assay, it was clear that he had isolated a high-affinity selective RAR that had no response to any other test ligand. Thus, without knowing any true direct target gene for retinoic acid, we were nonetheless able to isolate and characterize its receptor. Remarkably, Martin Petkovich in the Chambon lab isolated the same gene. Once again, this is an example where a new technique offered an entirely new approach to a problem. Both papers were published in the December 1987 issue of Nature (19, 20). With the combination of steroids, thyroid hormones, and vitamin A, the three elemental components of the nuclear receptor superfamily were in hand. By the time the RAR papers were published, Vincent with Na Yang, had already isolated two estrogen-related receptors termed ERR1 and 2 that would represent the first true orphan receptors in the evolving superfamily (21). A third receptor (ERR3) would be isolated 10 yr later (22). The three ERRs are distinguished by their ability to activate through ER response elements, but required no ligand. However, of potential major medical relevance, estrogen antagonists such as 4-hydroxy-tamoxifen silences ERR constitutive activity (23). The superfamily was growing exponentially, transforming into a new field, driven by a new breed of exceptional students and fellows attracted by the mechanics of transcription and its emerging link to physiology. For example, the RAR and TR offered an unprecedented look at understanding the action of vitamin A as a morphogen and the role of thyroxin in setting the basal metabolic rate of the body. We were a relatively small group, and our decision to work on multiple different receptor systems created a unique environment. Because there was so little overlap between projects, postdocs and students readily discussed all results, exchanged reagents and freely collaborated, resulting in a tremendous acceleration of progress. The high level of camaraderie was powered by the joie de vivre of the exciting discoveries and the ability of our family of students and postdocs to each adopt their own receptors. We all felt we were in a golden age and even more was to come.

In 1989, Jan Sap in Vennstrom’s group and Klaus Damm in our group demonstrated that the TR becomes oncogenic by mutation in the LBD (24, 25). Although we expected ligand-independent activation, it was clearly a constituitive repressor becoming the first example of a dominant-negative oncogene. The concept of the dominant-negative oncogene had been proposed one year earlier by Ira Herskowitz (26). This discovery changed our thinking on hormone action, and repression soon would be shown to be a common feature of receptor antagonists. David Mangelsdorf, who had arrived in the lab the year before was captivated by the glow of weakly hybridizing DNA bands and, in 1989, had amassed his own collection of orphan receptors, among which was the future retinoid X receptor (RXR) (27). In search for biological activity, a candidate ligand was found in lipid extracts from outdated human blood. However, the key test came from demonstrating that addition of all-trans retinoic acid to cultured cells would lead to its rapid metabolism coupled with the release of an inducing activity for RXR, which we termed retinoid X. David and his benchmate, Rich Heyman, began working on the chemistry of this inducer along with Gregor Eichele and Christine Thaller, then at Baylor College of Medicine (Houston, TX). This work led to the identification of 9-cis retinoic acid by our lab and a group at Hoffman LaRoche (Nutley, NJ) (28, 29). Like the retinal molecule in rhodopsin, 9-cis-retinoic acid represents the exploitation of retinoid isomerization by nature to control a key signaling pathway. More importantly, in the 39 yr since the discovery of aldosterone in 1953, this revelation would reawaken and reinvent the single most defining but dormant tool of endocrinology—ligand discovery. Indeed, the discovery that new receptors could lead to new ligands opened up an entirely new avenue of research. Like the puzzle of the structure of the benzene ring, which was solved in 1890 when Fredrick Kekule dreamed of a snake biting its own tail, the physiologic head of the “endocrine snake” and the molecular biology tail had come full circle. The era of reverse endocrinology was now upon us.

Response Elements: Deciphering The Scripts

One problem in addressing the downstream effects of our newly discovered receptors was that their response elements and target genes were by definition unknown. Kaz Umesono delved into this mystery and would produce a paradigm shift that would both solve the problem and further unify the field. With the view that the DBD functioned as a molecular receptor for its cognate hormone response element, meticulous mutational studies revealed two key DBD sequences, termed the P-box and D-box, that controlled the process (30).

The D-box was shown to direct dimerization, a feature previously thought to be unique to the LBD. One perplexing point was that the P-boxes of the nonsteroidal receptors were conserved, leading to the improbable prediction that many different receptors would recognize the same target sequence. By manual compilation and comparison of all known response elements, Kaz proposed a core hexamer— AGGTCA—as the prototypic common target sequence. By requiring the half-site to be an obligate hexamer an underlying pattern—the direct repeat—emerged. In the direct repeat paradigm, we proposed that half-site spacing, not sequence difference, was the key ingredient to distinguishing the response elements. The metric was referred to as the 3-4-5 rule (31). According to the rule, direct repeats of AGGTCA spaced by three nucleotides, would be a vitamin D response element (DR-3), the same repeat spaced by four nucleotides a thyroid hormone response element (DR-4), and the same repeat spaced by five nucleotides a vitamin A response element (DR-5). Eventually, all steps from 0–5 on the DR ladder would be filled (Fig. 2). The validity of this paradigm was ensured by a crystal structure solved in collaboration with Paul Sigler’s group at Yale (32). Indeed, of the remaining 40 nonsteroidal receptors, all but three can be demonstrated to have a preferred binding site within some component of the direct repeat ladder. Exceptions include SHP and DAX, which lack DBDs, and farnesoid X receptor (FXR) that binds to the ecdysone response element as a palindrome with zero spacing. Kaz’s insight, by drawing commonality from diversity, came to solve a problem that impacted on virtually every receptor. Remarkably, each new receptor in the superfamily could immediately be assigned a place on the ladder. The ladder also provided a simple means to conduct a ligand screening assay in absence of any knowledge of an endogenous target gene. Kaz’s ladder was a turbo charge for the field. The next major advance in the field was the discovery of the RXR heterodimer. Although we knew that retinoid and thyroid receptors required a nuclear competence factor for DNA binding, its identity was unknown. We tested RXR, but our initial experiments were flawed. Of the first four papers describing the discovery, that from Chambon’s lab was most elegant because they simply purified an activity to homogeneity to find RXR (33)! Rosenfeld was first to publish, and Ozato, Pfahl and Kliewer all concurred (34–37). Tony Oro and Pang Yao in our lab soon published that the ecdysone receptor functions as a heterodimer with ultraspiracle, the insect homolog of RXR (38, 39), revealing that the ancient origins of the heterodimer which arose before the divergence of vertebrates and invertebrates.

Reverse Endocrinology: Decoding Physiology

The orphan receptors would transform our view of endocrine physiology with unexpected links to toxicology, nutrition, cholesterol, and triglyceride metabolism as well as to a myriad of diseases including atherosclerosis, diabetes, and cancer. The three RXR isoforms formed the core with 14 heterodimer partners including the vitamin D receptor (VDR), TR/, and RAR//. The initial adopters of orphan receptors included Giguere, Mangelsdorf, Weinberger, Bruce Blumberg, Steve Kliewer, and Barry Forman. Barry unlocked the first secret to for peroxisome proliferator-activated receptor (PPAR) by identifying prostaglandin J2 (PGJ2) as a high-affinity ligand (40). The second step, in collaboration with Peter Tontonoz in Bruce Spiegleman’s lab, revealed that PGJ2 was adipogenic in cell lines and perhaps more importantly that the synthetic antidiabetic drug Troglitazone was a potent PPAR agonist (41). Similar work was conducted and published by Kliewer, who had now moved to Glaxo (42). By acquiring a ligand, a physiologic response, and a drug, PPAR was suddenly transported to the center of a physiologic cyclone that would spin into its own specialty field. Since 1995, more than 1000 papers (see PubMed) have been published on PPAR and its natural and synthetic ligands. This early work illuminated the molecular strategy of reverse endocrinology and the emerging importance of the orphan receptors in human disease and drug discovery. Cary returned to the lab for a sabbatical and, with Barry, demonstrated that FXR was responsive to farnesoids and other molecules in the mevalonate pathway. The findings by Mangelsdorf that liver X receptors (LXRs) bound oxysterols (43) and by Kliewer, Mangelsdorf, and Forman that FXR is a bile acid receptor (44–46) provided a whole new conceptual approach to cholesterol and triglyceride homeostasis. The steroid and xenobiotic receptors (SXR)/pregnane X receptor (PXR) (47–49) and the constituitive androstane receptor (CAR) (50) respond to xenobiotics to activate genes for P450 Fig. 2. Examples of Receptor Heterodimer Combinations that Fill the Direct Repeat (DR) Response Element Ladder from DR1 to DR5 Evans enzymes, conjugation and transport systems that detoxify drugs, foreign chemicals, and endogenous steroids. RXR and its associated heterodimeric partners quickly established a new branch of physiology, shedding its dependence on endocrine glands and allowing the body to decode signals from environmental toxins, dietary nutrients, and common metabolites of intermediary metabolism.



The human body is, after all a living machine, a complex device that consumes and uses energy to sustain itself, defend against predators, and ultimately reproduce. One might reasonably ask, “If the superfamily acts through a common molecular template, can the family as a whole be viewed as a functional entity?” In other words, is there yet some overarching principle that we have yet to grasp. . . and might this imaginary principle lie at the heart of systems physiology? Simply stated, what led to the evolution of integrated physiology as the functional output of the superfamily? One obvious speculation is survival. To survive, all organisms must be able to acquire, absorb, distribute, store, and use energy. The receptors are exquisitely evolved to manage fuel—everything from dietary and endogenous fats (PPARs), cholesterol (LXR, FXR), sugar mobilization (GR), salt (MR), and calcium (VDR) balance and maintenance of basal metabolic rate (TR). Because only a fraction of the material we voluntarily place in our bodies is nutritional, the xenobiotic receptors (PXR, CAR) are specialized to defend against the innumerable toxins in our environment. Survival also means reproduction, which is controlled by the gonadal steroid receptors (progesterone receptor, ER, AR). However, fertility is dependent on nutritional status, indicating the presumptive communication between these two branches of the family. The third key component managed by the nuclear receptor family is inflammation. During viral, bacterial, or fungal infection, the inflammatory response defends the body while suppressing appetite, conserving fuel, and encouraging sleep (associated with cytokine release). However, if needed, even an ill body is capable of defending itself by releasing adrenal steroids, mobilizing massive amounts of fuel, and transiently suppressing inflammation. In fact, clinically, (with the exception of hormone replacement) glucocorticoids are only used as antiinflammatory agents. Other receptors including the RARs, LXRs, PPAR and , and vitamin D receptor protect against inflammation. Thus, nature evolved within the structure of the receptor the combined ability to manage energy and inflammation, indicating the important duality between these two systems. In aggregate, this commonality between distinct physiologic branches suggests that the superfamily might be approached as an intact functional dynamic entity.

Historically, endocrinologists and geneticists rarely saw eye to eye. As I have indicated in this perspective article, the disciplines have now become united in a new subject—transcriptional physiology. With this in mind, we might expect the existence of larger organizational principles that establish how the various evolutionary branches of the superfamily integrate to form whole body physiology. The existence of molecular rules governing the function and evolution of a megagenetic entity like the nuclear receptor superfamily, if correct, may be useful in understanding complex human disease and provide a conceptual basis to create more effective pharmacology. With so much accomplished in the last 20 yr (see Fig. 3), there are glimpses of clarity—enough to see the enormity and wonder of the problem and enough to know there is still a long and challenging voyage ahead. But who knows, the next breakthrough may only be a stone’s throw away.


Pierre Chambon MD

Recipient of the Canada Gairdner International Award, 2010
“For the elucidation of fundamental mechanisms of transcription in animal cells and to the discovery of the nuclear receptor superfamily.”

Institut de Génétique et de Biologie Moléculaire et Cellulaire (IGBMC), Illkirch-Graffenstaden, France

Dr. Pierre Chambon is Honorary Professor at the College de France (Paris), and Emeritus Professor at the Faculté de Médecine of the Strasbourg University. He was the Founder and former Director of the IGBMC, and also the Founder and former Director of the Institut Clinique de la Souris (ICS/MCI), in Strasbourg.

Dr. Pierre Chambon is a world expert in the fields of gene structure, and transcriptional control of gene expression. During the last 25 years his studies on the structure and function of nuclear receptors has changed our concept of signal transduction and endocrinology. By cloning the estrogen and progesterone receptors, and discovering the retinoic acid receptor family, he markedly contributed to the discovery of the superfamily of nuclear receptors and to the elucidation of their universal mechanism of action that links transcription, physiology and pathology. Through extensive site-directed mutagenesis and genetic studies in the mouse, Pierre Chambon has unveiled the paramount importance of nuclear receptor signaling in embryonic development and homeostasis at the adult stage. The discoveries of Pierre Chambon have revolutionized the fields of development, endocrinology and metabolism, and their disorders, pointing to new tactics for drug discovery, and finding important applications in biotechnology and modern medicine.

These scientific achievements are logically inscribed in an uninterrupted series of discoveries made by Pierre Chambon over the last 45 years in the field of transcriptional control of gene expression in higher eukaryotes: discovery of PolyADPribose (1963), discovery of multiple RNA polymerases differently sensitive to a-amanitin (1969), contribution to elucidation of chromatin structure: the Nucleosome (1974), discovery of animal split genes (1977), discovery of enhancer elements (1981), discovery of multiple promoter elements and their cognate factors (1980-1993).

Pierre Chambon has received numerous international awards, including the 2004 Lasker Basic Medical Research Award for the discovery of the superfamily of nuclear hormone receptors and the elucidation of a unifying mechanism that regulates embryonic development and diverse metabolic pathways. He is a member of the French Académie des Sciences, and also a Foreign Member of the National Academy of Sciences (USA) and of the Royal Swedish Academy of Sciences. Pierre Chambon serves on a number of editorial boards, including Cell, and Molecular Cell. Pierre Chambon is author of more than 900 publications. He has been ranked fourth among most prominent life scientists for the 1983-2002 period.

An Interview with Pierre Chambon
2004 Albert Lasker Basic Medical Research Award

Pierre Chambon, MD

​Honorary Professor at the Collège-de-France and Professor of Molecular Biology and Genetics, Institute for Advanced Study, University of Strasbourg; Group Leader, Institut de Génétique et de Biologie Moléculaire et Cellulaire (IGBMC), Illkirch-Graffenstaden, Strasburg, France

A pioneer in the fields of gene structure and transcriptional control of gene expression, Dr. Chambon has fundamentally changed our understanding of signal transduction, which has led to revolutionary new tactics for drug discovery. His work elucidated how molecules that promote gene transcription are organized and regulated in eukaryotic organisms and, independently of Dr. Ronald Evans, he discovered in 1987 the retinoid receptor families, which led to the discovery and characterization of the superfamily of nuclear hormone receptors, including steroid and retinoid receptors.

Dr. Chambon’s previous research led to the discovery of PolyADPribose, multiple RNA polymerases differentially sensitive to α-amaniti, and has markedly contributed to the elucidation of the nucleosome and chromatin structure, as well as to the discovery of animal split genes, DNA sequences called enhancer elements, and multiple promoter elements and their cognate factors. These discoveries have greatly enhanced understanding of embryonic development and cell differentiation. To further studies of various nuclear receptors, Dr. Chambon has developed a method that allows in the mouse the generation of somatic mutations of any gene, at any time, and in any specific cell type, a tool valuable in generating mouse models of cancer.

In 1994, Dr. Chambon took on the role of founding a major research institute in France. As the first director of IGBMC, he built the institute to encompass hundreds of top researchers and multiple research programs funded by public agencies and private industry. In 2002, he founded and was the first director of the Institut Clinique de la Souris in Strasbourg. In these positions, he has succeeded in supporting and influencing a generation of scientists.

Career Highlights

​2010  Canada Gairdner International Award

2004  Albert Lasker Basic Medical Research Award

2003  Alfred P. Sloan, Jr., Prize, General Motors Cancer Foundation

1999  Louisa Gross Horwitz Prize, Columbia University

1998  Robert A. Welch Award in Chemistry

1991  Prix Louis-Jeantet de médecine, Fondation Louis-Jeantet

1990  Sir Hans Krebs Medal, Federation of European Biochemical Societies

1988  King Faisal International Prize for Science, King Faisal Foundation

1987  Harvey Prize, Technicon-Israel Institute of Technology



Minireviews In This Series:

Thematic Minireview Series on Nuclear Receptors in Biology and Diseases

Sohaib Khan and Jerry B Lingrel

Steroid Receptor Coactivator (SRC) Family: Masters of Systems Biology

Brian York and Bert W. O’Malley

Estrogen Signaling via Estrogen Receptor β

Chunyan Zhao, Karin Dahlman-Wright, and Jan-Åke Gustafsson

Small Molecule Inhibitors as Probes for Estrogen and Androgen Receptor Action

David J. Shapiro, Chengjian Mao, and Milu T Cherian

Cellular Processing of the Glucocorticoid Receptor Gene and Protein: New Mechanisms for Generating Tissue Specific Actions of Glucocorticoids

Robert H Oakley and John A Cidlowski

Endogenous Ligands for Nuclear Receptors: Digging Deeper

Michael Schupp and Mitchell A. Lazar




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Larry H. Bernstein, MD, FCAP, Curator

Leaders in Pharmaceutical Intervention

2010 Douglas L. ColemanJeffrey M. Friedman

Shaw Laureates 2009 Life Science and Medicine

Douglas L. Coleman (6 October 1931 – 16 April 2014) was a scientist and professor at The Jackson Laboratory, in Bar Harbor, Maine. His work predicted that the ob gene encoded the hormoneleptin,[1] later co-discovered in 1994 by Jeffrey Friedman, Rudolph Leibel and their research teams at Rockefeller University.[2][3][4][5][6][7][8] This work has had a major role in our understanding of the mechanisms regulating body weight and that cause of humanobesity.[9]

Coleman was born in Stratford, Ontario. He obtained his BS degree from McMaster University in 1954 and his PhD in Biochemistry from the University of Wisconsin in 1958. He was elected a member of the US National Academy of Sciences in 1998. He won the Shaw Prize in 2009,[10] the Albert Lasker Award for Basic Medical Research in 2010, the 2012 BBVA Foundation Frontiers of Knowledge Award in the Biomedicine category and the 2013 King Faisal International Prize for Medicine[11] jointly with Jeffrey M. Friedman[9] for the discovery of leptin.

The Genetics of Obesity

Winner of the  2013 KFIP Prize for  Medicine

Professor Douglas Coleman was born on October 5, 1931, in Stratford, Ontario, Canada. He obtained a B.Sc. in Chemistry in 1954 from McMaster University in Hamilton, Ontario, then went to the University of Wisconsin in Madison, WI, U.S.A., where he obtained M.S. and Ph.D. degrees in Biochemistry in 1956 and 1958, respectively. He served as a Research Assistant at the University of Wisconsin from 1954-1957 and as E.I. Dupont de Nemours Fellow from 1957-1958. He joined the Jackson Laboratory in Bar Harbor, ME, where he spent his entire career rising from Associate Staff Scientist In 1958 to Senior Staff Scientist in 1968. He also served as Assistant Director for Research from 1969-1970 and Interim Director  from 1975-1976. Upon his retirement in 1991, he was appointed Senior Staff Scientist Emeritus at Jackson. He was also consultant to the National Health Institutes, serving on the Metabolism Study Section from 1972-1974 and was frequently consulted on various other special study sections involving genetic diabetes, obesity and nutrition. He also served as Visiting Professor at the University of Geneva (1979-1980).

Professor Coleman’s research interests focus on biochemical genetics, regulation of metabolism, obesity, diabetes and hormone action. He is best known for his studies on the obesity-diabetes syndrome. He discovered the db gene, one of the two genes responsible for the genetic events regulating appetite control. He carried out a series of fundamental experiments with parabiotic mice which demonstrated the hormone-hormone receptor axis of leptin and the leptin receptor long before their discovery. The discoveries of Coleman and Friedman represent one of the most important biological breakthroughs in recent decades.

Professor Coleman received several prestigious awards and honors, including the Claude Bernard Medal by the European Diabetes Foundation in 1977, the Distinguished Alumni Award in Science by McMaster University in 1999, the Gairdner International Award in 2005, the Shaw Prize for Life Sciences and Medicine in 2009 (jointly with Jeffrey M. Friedman), the Albert Lasker Basic Medical Research Award (jointly with Jeffrey M. Friedman) and the Outstanding Forest Stewardship Award (Maine Forest Service). He was elected to the National Academy of Sciences in 1991, and was awarded Honorary D.Sc. from Louisiana State University in 2005 and Honorary D.Sc. from McMaster University in 2006. He is a member of the American Association of Biological Chemists.

Professor Douglas Leonard Coleman was awarded the prize because the research findings by him and Professor Friedman led to the identification and characterization of the leptin pathway. This seminal discovery has had a major impact on our understanding of the biology of obesity, describing some of the key afferent pathways in body weight regulation active in man. Their fundamental discoveries have also helped in the recognition of more illuminating views of the endocrine system. Because of their major contribution to the field of the genetics of obesity they have been awarded King Faisal International Prize in Medicine for the year 2013.

Leaping for leptin: the 2010 Albert Lasker Basic Medical Research Award goes to Douglas Coleman and Jeffrey M. Friedman

Ushma S. Neill

J Clin Invest. 2010 Oct 1; 120(10): 3413–3418.
Published online 2010 Sep 21. doi:  10.1172/JCI45094

Douglas Coleman never intended to study diabetes or obesity. Jeffrey M. Friedman had childhood dreams of being a veterinarian. But together, the two scientists have opened the field of obesity research to molecular exploration. On September 21, the Albert and Mary Lasker Foundation announced that they will award Coleman and Friedman (Figure (Figure1)1) with the 2010 Albert Lasker Basic Medical Research Award in recognition of their contributions toward the discovery of leptin, a hormone that regulates appetite and body weight. This hormone provides a key means by which changes in nutritional state are sensed and in turn modulate the function of many other physiologic processes. The story of the discovery of the first molecular target of obesity is one of tenacity and determination.

Figure 1

Douglas Coleman (left) and Jeffrey M. Friedman (right) share the 2010 Albert Lasker Basic Medical Research Award for the discovery of leptin, a breakthrough that opened obesity research to molecular exploration.

From Canada to Maine

Douglas Coleman was raised in Ontario, Canada, the only child of English immigrant parents, who encouraged him to excel in school; he recalled, “Although my parents never had the luxury of completing high school, they always encouraged me to pursue a higher education, and in high school, I developed a keen interest in chemistry and biology.” Coleman pursued his interest in chemistry at McMaster University. It was there he met his future wife, Beverly Benallick, “the only girl to graduate in Chemistry in the Class of 1954.” During his time at McMaster University, Coleman began to focus on organic chemistry and had the fortune of working with, “a very dynamic professor, Sam Kirkwood, who not only taught me the rudiments of biochemistry, but also instilled an appreciation of the scientific method.” Kirkwood encouraged Coleman to continue his biochemistry studies at the University of Wisconsin, at which he received a PhD in 1958.

In those days, postdoctoral fellowships were rare, and graduates had two options: academia or industry. Coleman took a third option, as an associate staff scientist at what was then known as the Roscoe B. Jackson Memorial Laboratory in Bar Harbor, Maine. Coleman has noted, “My intention was to stay one or two years, expanding my skills in multiple fields, especially genetics and immunology. To my great pleasure, The Jackson Laboratory provided a rich environment, including world-class animal models of disease, interactive colleagues, and a backyard that included the stunning beauty of Acadia National Park.” The Coleman family put down roots, raising their three sons there as Coleman rose through the ranks to senior staff scientist and served terms as assistant director of research and interim director (Figure (Figure2).2). He noted, “Without a doubt, I was lucky in my choice of starting my career at The Jackson Laboratory. It was a wonderful place in which to work, and I never pursued another position.”

Figure 2

Coleman at the bench at The Jackson Laboratory in 1960.

Making magic from a mutant

His early work involved muscular dystrophy and the development of a new field, mammalian biochemical genetics, establishing that genes control enzyme turnover as well as structure. However, his focus changed when a colleague asked for his help characterizing a mutant (Figure (Figure3)3) that had spontaneously arisen at the labs. He recalled, “Initially, I had no intention of studying the diabetes/obesity syndrome, but in 1965, a spontaneous mouse mutation was discovered, and I began research that would consume much of my scientific thought for the better part of three decades.” The new mutant was polydipsic and polyuric as well as being massively obese and hyperphagic. His colleague, Katherine Hummel, was studying diabetes insipidus and asked if he could determine whether the new mutant had diabetes insipidus or mellitus. He reported back that it was diabetes mellitus: “Her initial response was that she was not interested, but I convinced her that with a little further work we could produce a solid manuscript announcing this potentially valuable mutant to the world.” This mouse owed its phenotype to two defective copies of a gene that researchers dubbed diabetes (db) (1).

Figure 3

Wild-type and obese mice.

When Coleman and his colleagues began characterizing the db/db mouse, they began to ponder whether some circulating factor might regulate the severity of diabetes: perhaps a factor in the normal mouse could inhibit the development of the obesity and diabetes found in the db/db mutant. Conversely, perhaps a circulating factor present in the db/db mouse might cause the diabetes-like syndrome in the normal mouse. If the hypothetical factor was carried through the blood, Coleman reasoned, they could test for its presence by linking the blood supplies of the various mouse strains — an experimental setup called parabiosis. Fortunately, others at The Jackson Laboratory were using parabiosis to assess whether any circulating factors were involved in anemic mutants, and they were able to show Coleman how to do it successfully.

When Coleman hooked the wild-type mice and the db/db mice together, rather than overeating, as the db/dbmice did, the wild-type mice stopped eating and died from starvation (Figure (Figure44 and ref. 2). His hypothesis was correct: the db/db mice indeed must have released a factor that inhibited the wild-type animals’ drive to eat, but the mutant animals could not respond to it.

Figure 4

Summary of parabiosis experiments performed by Coleman.

Coleman needed more proof of this mystery circulating factor regulating food intake. He turned to another overweight mouse that also had arisen by chance at The Jackson Laboratory, this one called “obese,” whose aberrant physiology arises from two defective copies of a different gene (ob) (3). Unfortunately, the ob/obmouse was on a different genetic background, and due to immune-mediated rejection, parabiosis could only be performed successfully on mice with the same strain background. Coleman described his need for resolve, “Since the obese and diabetic mutants were on different genetic backgrounds, it took years for me to be able to perform all of the desired pairings.”

Coleman persevered and finally got the strains to match so he could successfully hook them together in a parabiosis experiment. When joined to a db/db mouse, the ob/ob mouse stopped eating and starved to death, while the db/db mouse remained obese, just as the normal mice had in the previous experiment. In contrast, attaching wild-type mice to ob/ob animals did nothing to the wild-type mice and caused the ob/ob mice to limit their food consumption and gain less weight (Figure (Figure4).4). Coleman concluded that the ob/ob mice failed to produce a hormone that inhibits eating, while the db/db mice overproduced it but lack the receptor to transmit the hormonal signal (4).

Coleman faced some skepticism for his conclusion that obesity was not just about willpower and eating habits but also involved chemical and genetic factors. In this regard, he said, “When I published these findings, the long-standing dogma was that obesity was a behavioral problem (a lack of willpower) and not a physiological problem (a hormonal imbalance). I had to deal with this behavioral dogma most of my career.”

To validate his hypothesis, Coleman would need to identify the db and ob genes and protein products, a task that proved to be an insurmountable challenge at the time. He noted, “Definitive proof of my conclusions required isolating the satiety factor — a feat that resisted rigorous experimentation.” That is, until Jeffrey Friedman set his sights upon the task.

 After his third year of internal medicine residency at Albany Medical Center Hospital, Friedman  had no concrete plans for the following year, as he was not scheduled to begin a fellowship at the Brigham and Women’s Hospital in Boston until a year later. Friedman recalled, “I had no particular plans for the gap year, and John Balint, one of my professors, thought I might like research — why he thought I might have some particular aptitude, I can’t really tell. He said, ‘I have this friend at Rockefeller [Mary Jeanne Kreek], why don’t you go spend a year with her and see if you like research?’ I didn’t know what else I was going to do. My mother thought I should go spend the year as a ship’s doctor.”
A fat chance

Friedman was enraptured by what Kreek studied: how molecules control behavior. “That was 1981 and it was beginning to be evident that molecular biology was going to have a big impact, so instead of going to the Brigham for a fellowship, I abandoned medicine and decided to get a PhD with Jim Darnell [2002 Lasker award winner for his work in RNA processing and cytokine signaling], who was one of the leaders in molecular biology,” he noted. Friedman’s thesis was on the regulation of liver gene expression — how genes are turned on and off as liver regenerates. However, there was something he did on the side that was more impactful: Kreek had asked him to work with Bruce Schneider, another faculty member at Rockefeller University, to make an RIA for β-endorphin. However, Schneider’s primary interest was not in β-endorphin, but rather in cholecystokinin (CCK). In 1979, Rosalyn Yalow had published a paper in which she reported reduced levels of CCK in the brain of ob/ob mice and boldly claimed that CCK was the circulating factor that caused the ob/ob mice to be fat (5). Friedman recalled, “Well, Bruce had the exact opposite data, this was published in the JCI (6), and this started a battle with Yalow over who was correct. To address this, in 1982 Don Powell, Bruce, and I set out to clone the Cck gene so we could map it. We collaborated with Peter D’Eustachio at NYU, who showed that it was on chromosome 9 (7); ob is on 6, db is on 4. I still have Peter’s notebook entry from that time in which he wrote, ‘CCK does not map to chromosome 6, home ofob.’” So the question for Friedman became, if the circulating hormone is not CCK, then what is? When he started his own laboratory in 1986 at Rockefeller, he set out to find it, and as he recalls, “In a way what theob mouse represented to me was another instance where a molecule was controlling a behavior, the same as in Mary Jeanne’s lab.”

Do these genes make me look fat?

In the mid ’80s, positional cloning was not easy, but Friedman turned to the then-new techniques of physical gene mapping, complimented by conventional genetic mapping in mice. It had long been known that the obgene resided somewhere on mouse chromosome 6, but narrowing down the region was arduous, as the trait is recessive, necessitating the breeding of several generations. Friedman and his laboratory first determined which DNA markers were inherited along with the obese phenotype in over 1,600 mice crossbred from obese and nonobese strains. He remembers, “It was a mind numbing exercise you hoped someday would lead somewhere.” Since the genetic and physical maps are colinear, DNA markers that were linked to ob in genetic crosses could be used to clone the surrounding DNA. Using this approach, they eventually identified the portion of the genome in which all markers were always coinherited with ob among the progeny of the crosses. This region defined the chromosomal region in which the ob gene resided. As they had predicted when the crosses were set up, this region corresponded to an approximately 300,000–base pair region on chromosome 6. They then screened recombinant clones across this region for exon-intron boundaries, which indicate the presence of genes. One of the first three genes they isolated was expressed exclusively in adipose tissue, and the expression of the mutant gene was found to be 20 times greater in one of the ob/ob mutants than in controls. In a second mutant, the gene was not expressed at all, providing clear evidence that this gene encoded the ob gene. When they looked in the human genome, they found an ob homolog that was 84% identical with the mouse ob gene, establishing ob as a highly conserved, biologically important gene (8).

Once a fat-specific gene was found in the vicinity of ob, he remembered being almost numb with excitement as a set of confirmatory experiments unfolded. “I went in late on a Saturday night, and I found a radioactive probe for this gene, and I found a blot with RNA from fat tissue of normal and mutant mice. I hybridized the blot that evening and washed it at 1 in the morning. I couldn’t sleep, and I woke up at 5 or 6 and developed the blot. When I looked at the data, I immediately knew that we had cloned ob. When I saw it, I was in the darkroom, and I pulled up the film and looked at it under the light and got weak-kneed. I sort of fell backwards against the wall. This gene was in the right region of the chromosome, it was fat specific, and its expression was altered in two independent strains of ob mice. Before this, we didn’t know where ob would be expressed — and while fat was one of the tissues I considered, in principle the gene could have been expressed in any specialized cell type anywhere that had no obvious relationship to fat. But on the other hand, seeing a gene in the right region expressed exclusively in the fat . . . that gets your attention.” When he found out at 6 in the morning, he called his wife and said “we did it!,” and then, a few hours later he, called his former PhD advisor Jim Darnell: “I told him but I wasn’t sure he believed me.” That afternoon, he met some friends at Pete’s Tavern, “and we opened a bottle of champagne, and I told them, ‘I think this is going to be pretty big.’”

Next Friedman set his sights on actually identifying the product secreted by the ob gene and validating Coleman’s circulating hormone hypothesis. Together with Stephen Burley, his laboratory engineered E. colito fabricate the secreted protein, generated antibodies that would bind it, and showed that humans and rodents secrete it. In the last sentence of the 1995 Science paper describing these findings, Friedman “propose[d] that this 16-KD protein be called leptin, derived from the Greek root leptos, meaning thin” (9). The paper also showed that db/db mice made excess quantities of leptin, as predicted by Coleman, and its levels in plasma decreased in normal animals and obese humans after weight loss. He remembered, “It was an unbelievable time in the lab. The idea that there was this hormone that regulated body weight, and that we had found it, was just unimaginable. I’d wake up in the middle of the night just smiling.”

As for the name leptin, it has not only a Greek root, but a French one too. At a meeting, Friedman met Frenchman Roger Guillemin, who won a Nobel Prize for his work on peptide hormone production by the brain. A few weeks after the meeting, Friedman got a letter from him that he recalls saying, “I really liked what you had to say, but I have one quibble: you refer to these as obesity genes, but I think they are lean genes because the normal allele keeps you thin. But calling them lean genes sounds awkward. The nicest sounding root for thin is from Greek, so I propose you call ob and db ‘lepto-genes.’” So when it came time to name it, Friedman remembered Guillemin’s suggestion, and therein, the name leptin was coined.

Leptin’s legacy

Later in 1995, another group described the leptin receptor (10), and then subsequently, Friedman and another group showed that this leptin receptor is encoded by the db gene and has multiple forms, one of which is defective in Coleman’s originally described db/db mice (11, 12). Friedman also showed that the leptin receptor is especially abundant in the hypothalamus in which leptin can activate signal transduction and phosphorylation of the Stat3 transcription factor (13).

Over the years, numerous laboratories have studied leptin’s mechanism of action. Leptin acts on receptors expressed in groups of neurons in the hypothalamus, in which it inhibits appetite, in part, by counteracting the effects of neuropeptide Y, a potent feeding stimulant secreted by cells in the gut and in the hypothalamus, by thwarting the effects of anandamide, another potent feeding stimulant, and by promoting the synthesis of α-MSH (melanocyte stimulating hormone), an appetite suppressant (14). Leptin is produced in large amounts by white adipose tissue but can also be produced in lesser amounts by brown adipose tissue, syncytiotrophoblasts, ovaries, skeletal muscle, stomach, mammary epithelial cells, bone marrow, pituitary, and liver. Leptin’s actions are also not limited to regulating food intake, as it is has been shown to have roles in fertility, immunity, angiogenesis, and surfactant production. Friedman adds that the hormone, “has effects on many physiological systems, including the immune system where it modulates T cells, macrophages, and platelets. It now appears that leptin provides a key means by which nutritional state can regulate a host of other physiological systems.” While most of these actions are mediated by effects on the CNS, two of many key questions are, which of leptin’s effects on peripheral systems are direct, and which are indirect via the brain?

A magic bullet?

The first proof that leptin was important in humans came in 1997 when Stephen O’Rahilly and colleagues found two morbidly obese children who carried a mutation in the leptin gene (15). These researchers went on to show that leptin-replacement therapy could be useful in individuals with leptin mutations (16). Injection of leptin into these children led to rapid weight loss and markedly reduced food intake (Figure (Figure5).5). Leptin-replacement therapy also has potent effects in other clinical settings, including lipodystrophy, a disease state in which animals and humans have little white fat and develop severe diabetes, with profound insulin resistance and high plasma lipid levels. Because this syndrome is associated with low circulating levels of leptin, Shimomura and colleagues tested the effects of leptin-replacement therapy in mice and showed that it was highly effective (17); similar efficacy was later shown in humans (18). More recently, leptin treatment has shown a profound anti-diabetic effect in type 1 diabetic animals (19). Leptin replacement has also been shown to be of clinical benefit in other states of leptin deficiency, including hypothalamic amenorrhea (20).

Figure 5

Effects of r-metHuLeptin on the weight a child with congenital leptin deficiency.

Excited by leptin’s potential for the treatment of obesity, the biotech company Amgen paid $20 million to Rockefeller to license the hormone. With so much of the world’s population overweight or obese, a treatment or cure would be a major advance in public health and would likely be very lucrative. Amgen sponsored a large clinical trial, giving leptin to overweight adults, but while a subset of obese patients lost significant amounts of weight on leptin, the average magnitude of the effect was minimal, dampening hopes that leptin was the magic bullet in the obesity fight (21). After the trial, Amgen announced that they had suspended studies of the effects of leptin for the treatment of human obesity.

Friedman says he understands why the trials failed: “Even before leptin was tested in obese patients, we knew from animal studies that this hormone was not likely to be a panacea for every obese patient and that the response seen in ob/ob mice wasn’t going to be the typical case for obese humans. Leptin levels are elevated in obese humans, suggesting that obesity is often associated with leptin resistance and raising the possibility that increasing already high levels was going to be of arguable benefit.” The key to making leptin work may be in coaxing the brain to respond to leptin: some people are simply not sensitive enough or they develop resistance. Friedman predicts that through personalized medicine, doctors may at some point be able to identify which obese people will respond to leptin. In the meantime, there is some clinical evidence that leptin’s ability to reduce weight among obese patients can be restored by combining it with other agents (22).

The thrill of discovery

For all the social implications, potential profits, and medical possibilities, Friedman is circumspect but proud about the discovery of leptin, saying, “whether it finds its way into general usage as an antiobesity drug, the use of modern methods to identify and target the components of the leptin- signaling pathway will, I believe, form the basis for new pharmacological approaches to the treatment of obesity and other nutritional disorders.” Coleman agrees, stating that “with the discovery of leptin and the subsequent cloning of the leptin receptor, the field exploded. With these findings, two long-standing misconceptions were definitively laid to rest: obesity was not merely a behavioral problem but rather had a significant physiological component; and adipose tissue was not merely a fat-storage site but rather an important endocrine organ.”

Both Coleman and Friedman (Figure (Figure6)6) were overwhelmed and humbled by the news that they would receive the 2010 Lasker Award for Basic Medical Research. Coleman notes, “I have always viewed this award as one of the most esteemed of the several truly prestigious biomedical research awards, and it is with great pride and humility that I accept this prestigious prize. I was also especially delighted to learn that I would be sharing this award with Jeffrey Friedman, who always acknowledged my earlier contributions to our field.” Friedman added, “It is an honor to join a group of other winners who really are at the highest level of science. To be placed among them is just hard to fathom.”

Figure 6

Coleman and Friedman, together at The Jackson Laboratory, in 1995.

Coleman retired from his scientific career in 1991. He has said that at his retirement ceremony “someone commented that my career was characterized by the ability to use the simplest technique to answer the most complex biological questions.” Friedman, however, is still at the bench and active as ever in his hunt to determine exactly how leptin regulates food intake. Through their determination and persistence, the two have provided a molecular framework for understanding obesity, but they have different opinions about how much luck played into their findings. Coleman has noted that he favors the Louis Pasteur quote, “Luck favors the prepared mind.” But Friedman has a different perspective, stating “my story suggests that in many cases, the prepared mind is favored by chance.”


As Coleman was away and unavailable for comment during the preparation of this article, his quotations were taken from an autobiography he wrote when accepting the Shaw prize in 2009, from his acceptance remarks for the Lasker prize, and from a profile written by Luther Young posted on the Bangor Daily Newsin 2009 (,118612?print=1).

1. Hummel KP, Dickie MM, Coleman DL. Diabetes, a new mutation in the mouse. Science.1966;153(740):1127–1128. doi: 10.1126/science.153.3740.1127. [PubMed] [Cross Ref]
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3. Ingalls AM, Dickie MM, Snell GD. Obese, a new mutation in the house mouse. J Hered.1950;41(12):317–318. [PubMed]
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5. Straus E, Yalow RS. Gastrointestinal peptides in the brain. Fed Proc. 1979;38(9):2320–2324. [PubMed]
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7. Friedman JM, Schneider BS, Barton DE, Francke U. Level of expression and chromosome mapping of the mouse cholecystokinin gene: implications for murine models of genetic obesity. Genomics.1989;5(3):463–469. doi: 10.1016/0888-7543(89)90010-4. [PubMed] [Cross Ref]
8. Zhang Y, Proenca R, Maffei M, Barone M, Leopold L, Friedman JM. Positional cloning of the mouse obese gene and its human homologue. Nature. 1994;372(6505):425–432. doi: 10.1038/372425a0. [PubMed][Cross Ref]
9. Halaas JL, et al. Weight-reducing effects of the plasma protein encoded by the obese gene. Science.1995;269(5223):543–546. doi: 10.1126/science.7624777. [PubMed] [Cross Ref]
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11. Chen H, et al. Evidence that the diabetes gene encodes the leptin receptor: identification of a mutation in the leptin receptor gene in db/db mice. Cell. 1996;84(3):491–495. doi: 10.1016/S0092-8674(00)81294-5.[PubMed] [Cross Ref]
12. Lee GH, et al. Abnormal splicing of the leptin receptor in diabetic mice. Nature. 1996;379(6566):632–635. doi: 10.1038/379632a0. [PubMed] [Cross Ref]
13. Vaisse C, Halaas JL, Horvath CM, Darnell JE, Jr, Stoffel M, Friedman JM. Leptin activation of Stat3 in the hypothalamus of wild-type and ob/ob mice but not db/db mice. Nat Genet. 1996;14(1):95–97. [PubMed]
14. Friedman JM, Halaas JL. Leptin and the regulation of body weight in mammals. Nature.1998;395(6704):763–770. doi: 10.1038/27376. [PubMed] [Cross Ref]
15. Montague CT, et al. Congenital leptin deficiency is associated with severe early-onset obesity in humans.Nature. 1997;387(6636):903–908. doi: 10.1038/43185. [PubMed] [Cross Ref]
16. Farooqi IS, et al. Beneficial effects of leptin on obesity, T cell hyporesponsiveness, and neuroendocrine/metabolic dysfunction of human congenital leptin deficiency. J Clin Invest.2002;110(8):1093–1103. [PMC free article] [PubMed]
17. Shimomura I, Hammer RE, Ikemoto S, Brown MS, Goldstein JL. Leptin reverses insulin resistance and diabetes mellitus in mice with congenital lipodystrophy. Nature. 1999;401(6748):73–76. doi: 10.1038/43448.[PubMed] [Cross Ref]
18. Oral EA, et al. Leptin-replacement therapy for lipodystrophy. N Engl J Med. 2002;346(8):570–578. doi: 10.1056/NEJMoa012437. [PubMed] [Cross Ref]
19. Wang MY, et al. Leptin therapy in insulin-deficient type I diabetes. Proc Natl Acad Sci U S A.2010;107(11):4813–4819. doi: 10.1073/pnas.0909422107. [PMC free article] [PubMed] [Cross Ref]
20. Welt CK, et al. Recombinant human leptin in women with hypothalamic amenorrhea. N Engl J Med.2004;351(10):987–997. doi: 10.1056/NEJMoa040388. [PubMed] [Cross Ref]
21. Heymsfield SB, et al. Recombinant leptin for weight loss in obese and lean adults: a randomized, controlled, dose-escalation trial. JAMA. 1999;282(16):1568–1575. doi: 10.1001/jama.282.16.1568.[PubMed] [Cross Ref]
22. Roth JD, et al. Leptin responsiveness restored by amylin agonism in diet-induced obesity: evidence from nonclinical and clinical studies. Proc Natl Acad Sci U S A. 2008;105(20):7257–7262. doi: 10.1073/pnas.0706473105. [PMC free article] [PubMed] [Cross Ref]
Autobiography of Jeffrey M Friedman

My laboratory identified leptin, a hormone that is produced by fat tissue. Leptin acts on the brain to modulate food intake and functions as an afferent signal in a feedback loop that regulates weight. My route to this hormone is filled with a number of chance events and turns of fate that were in no way predictable at the time that I started my career.I grew up in the suburbs of New York City in a village where children had enormous freedom. I recall from an early age riding my bicycle everywhere without my parents, or anyone else for that matter, knowing my whereabouts. My father was a radiologist and my mother was a teacher. No one in my family or community had pursued an academic career and at the time I was completely unaware of the possibility that one could make a career in science. In my family, the highest level of achievement was to become a doctor and, despite my earliest dreams of a career as a professional athlete (made unlikely by a notable lack of talent) and a later wish to become a veterinarian, I became a doctor.I was originally trained in internal medicine with some subspecialty training in gastroenterology. In medical school and as a medical resident, I participated in some modest research studies. The first piece of work I completed related to the effects of dietary salt on the regulation of blood pressure. After completing this project, I excitedly submitted a paper for publication. I remember one of the reviews verbatim: “This paper should not be published in the Journal of Clinical Investigation or anywhere else.” Fortunately, one of my mentors in medical school still thought I might have some aptitude for research. He suggested that I go to The Rockefeller University to work in a basic science research laboratory. I joined the laboratory of Dr Mary-Jeanne Kreek to study the effects of endorphins in the development of narcotic addiction.I was fascinated by the idea that endogenous molecules could alter behaviour and emotional state. At The Rockefeller University, I met another scientist, Bruce Schneider. Bruce was studying cholecystokinin (CCK), a peptide hormone that is secreted by intestinal cells. CCK aids digestion by stimulating the secretion of enzymes from the pancreas and bile from the gallbladder. CCK had also been found in neurons of the brain, although its function there was less clear. In the late 1970s, it was shown that injections of CCK reduce food intake. This finding appealed to me as another example of how a single molecule can change behavior. One other fact also piqued my interest: There were indications that the levels of CCK were decreased in a genetically obese ob/ob mouse. These mutant mice are massively obese as a consequence of a defect in a single gene. The mice eat excessively and weigh 3 to 5 times as much as normal mice. It was thus hypothesized that CCK functions as an endogenous appetite suppressant and that a deficiency of CCK caused the obesity evident in ob/ob mice. Fascinated by this possibility, I set out to establish the possible role of CCK in the pathogenesis of obesity in these animals. To do this I was going to need additional training in basic research, so I abandoned my plans to continue medical training in gastroenterology and instead entered the PhD program at The Rockefeller University.As a PhD student I worked in the laboratory of Jim Darnell, studying the regulation of gene expression in liver, and learning the basic tools of molecular biology. But I carried my interest in the ob/ob gene with me. At the end of my graduate studies, two colleagues and I successfully isolated the CCK gene from mouse. One of the first studies we performed after isolating the gene was to determine its chromosomal position. We found that the CCK gene was not on chromosome 6, where the ob mutation had been localized, which thus excluded defective CCK as the cause of the obesity. The question thus remained: What is the nature of the defective gene in ob/ob mice?

After receiving my PhD in 1986, I became an assistant professor at The Rockefeller University and set out to answer this question. The culmination of what proved to be an 8-year odyssey was the identification of the ob gene in 1994. We now know that the ob gene encodes the hormone leptin. The discovery of this hormone, a singular event in my life, was absolutely exhilarating. The realization that nature had happened upon such a simple and elegant solution for regulating weight was the closest thing I have ever had to a religious experience. Subsequent studies revealed that injections of leptin dramatically decrease the food intake of mice and other mammals. My current studies now focus on several questions, including the one that originally aroused my interest in this mutation: How is it that a single molecule – leptin – profoundly influences feeding behavior? An esteemed colleague of mine remarked recently that I had searched for the ob gene primarily so that I could approach the question I had started with. It is as yet unclear whether I will succeed in understanding how a single molecule can influence a complex behaviour.

  1. Coleman, DL (1978). “Obese and Diabetes: two mutant genes causing diabetes-obesity syndromes in mice”. Diabetologia 14: 141–148. doi:10.1007/bf00429772.
  2. Jump up^ Green ED, Maffei M, Braden VV, Proenca R, DeSilva U, Zhang Y, Chua SC Jr, Leibel RL, Weissenbach J, Friedman JM. (August 1995). “The human obese (OB) gene: RNA expression pattern and mapping on the physical, cytogenetic, and genetic maps of chromosome 7”.Genome Research 5 (1): 5–12. doi:10.1101/gr.5.1.5.PMID 8717050.
  3. Jump up^ Shell E (January 1, 2002). “Chapter 4: On the Cutting Edge”. The Hungry Gene: The Inside Story of the Obesity Industry. Atlantic Monthly Press. ISBN 978-1-4223-5243-4.
  4. Jump up^ Shell E (January 1, 2002). “Chapter 5: Hunger”. The Hungry Gene: The Inside Story of the Obesity Industry. Atlantic Monthly Press.ISBN 978-1-4223-5243-4.
  5. Jump up^ Zhang Y, Proenca R, Maffei M, Barone M, Leopold L, Friedman JM (December 1994). “Positional cloning of the mouse obese gene and its human homologue”. Nature 372 (6505): 425–432.doi:10.1038/372425a0. PMID 7984236.
  6. Jump up^ Rosenbaum M (1998). “Leptin”. The Scientist Magazine.
  7. Jump up^ Okie S (February 11, 2005). “Chapter 2: Obese Twins and Thrifty Genes”. Fed Up!: Winning the War Against Childhood Obesity. Joseph Henry Press, an imprint of the National Academies Press. ISBN 978-0-309-09310-1.
  8. Jump up^ Zhang, Y; Proenca, P; Maffei, M; Barone, M; Leopold, L; Friedman, JM. (1994). “Positional cloning of the mouse obese gene and its human homologue”. Nature 372 (6505): 425–432.doi:10.1038/372425a0. PMID 7984236.
  9. ^ Jump up to:a b Friedman, Jeffrey (2014). “Douglas Coleman (1931–2014) Biochemist who revealed biology behind obesity”. Nature 509 (7502): 564. doi:10.1038/509564a. PMID 24870535.
  10. Jump up^ Shaw Prize 2009
  11. Jump up^ King Faisal Prize 2013 for Medicine

A Metabolic Master Switch Underlying Human Obesity

Researchers find pathway that controls metabolism by prompting fat cells to store or burn fat

Aug 21, 2015

Researchers find pathway that controls metabolism by prompting fat cells to store or burn fat.

Obesity is one of the biggest public health challenges of the 21st century. Affecting more than 500 million people worldwide, obesity costs at least $200 billion each year in the United States alone, and contributes to potentially fatal disorders such as cardiovascular disease, type 2 diabetes, and cancer.

But there may now be a new approach to prevent and even cure obesity, thanks to a study led by researchers at MIT and Harvard Medical School. By analyzing the cellular circuitry underlying the strongest genetic association with obesity, the researchers have unveiled a new pathway that controls human metabolism by prompting our adipocytes, or fat cells, to store fat or burn it away.

“Obesity has traditionally been seen as the result of an imbalance between the amount of food we eat and how much we exercise, but this view ignores the contribution of genetics to each individual’s metabolism,” says senior author Manolis Kellis, a professor of computer science and a member of MIT’s Computer Science and Artificial Intelligence Laboratory (CSAIL) and of the Broad Institute.

New mechanism found

The strongest association with obesity resides in a gene region known as “FTO,” which has been the focus of intense scrutiny since its discovery in 2007. However, previous studies have failed to find a mechanism to explain how genetic differences in the region lead to obesity.

“Many studies attempted to link the FTO region with brain circuits that control appetite or propensity to exercise,” says first author Melina Claussnitzer, a visiting professor at CSAIL and instructor in medicine at Beth Israel Deaconess Medical Center and Harvard Medical School. “Our results indicate that the obesity-associated region acts primarily in adipocyte progenitor cells in a brain-independent way.”

To recognize the cell types where the obesity-associated region may act, the researchers used annotations of genomic control switches across more than 100 tissues and cell types. They found evidence of a major control switchboard in human adipocyte progenitor cells, suggesting that genetic differences may affect the functioning of human fat stores.

To study the effects of genetic differences in adipocytes, the researchers gathered adipose samples from healthy Europeans carrying either the risk or the non-risk version of the region. They found that the risk version activated a major control region in adipocyte progenitor cells, which turned on two distant genes, IRX3 and IRX5.

Control of thermogenesis

Follow-up experiments showed that IRX3 and IRX5 act as master controllers of a process known as thermogenesis, whereby adipocytes dissipate energy as heat, instead of storing it as fat. Thermogenesis can be triggered by exercise, diet, or exposure to cold, and occurs both in mitochondria-rich brown adipocytes that are developmentally related to muscle, and in beige adipocytes that are instead related to energy-storing white adipocytes.

“Early studies of thermogenesis focused primarily on brown fat, which plays a major role in mice, but is virtually nonexistent in human adults,” Claussnitzer says. “This new pathway controls thermogenesis in the more abundant white fat stores instead, and its genetic association with obesity indicates it affects global energy balance in humans.”

The researchers predicted that a genetic difference of only one nucleotide is responsible for the obesity association. In risk individuals, a thymine (T) is replaced by a cytosine (C) nucleobase, which disrupts repression of the control region and turns on IRX3 and IRX5. This then turns off thermogenesis, leading to lipid accumulation and ultimately obesity.

By editing a single nucleotide position using the CRISPR/Cas9 system — a technology that allows researchers to make precise changes to a DNA sequence — the researchers could switch between lean and obese signatures in human pre-adipocytes. Switching the C to a T in risk individuals turned off IRX3 and IRX5, restored thermogenesis to non-risk levels, and switched off lipid storage genes.

“Knowing the causal variant underlying the obesity association may allow somatic genome editing as a therapeutic avenue for individuals carrying the risk allele,” Kellis says. “But more importantly, the uncovered cellular circuits may allow us to dial a metabolic master switch for both risk and non-risk individuals, as a means to counter environmental, lifestyle, or genetic contributors to obesity.”

Success in human and mouse cells

The researchers showed that they could indeed manipulate this new pathway to reverse the signatures of obesity in both human cells and mice.

In primary adipose cells from either risk or non-risk individuals, altering the expression of either IRX3 or IRX5 switched between energy-storing white adipocyte functions and energy-burning beige adipocyte functions.

Similarly, repression of IRX3 in mouse adipocytes led to dramatic changes in whole-body energy balance, resulting in a reduction of body weight and all major fat stores, and complete resistance to a high-fat diet.

“By manipulating this new pathway, we could switch between energy storage and energy dissipation programs at both the cellular and the organismal level, providing new hope for a cure against obesity,” Kellis says.

The researchers are currently establishing collaborations in academia and industry to translate their findings into obesity therapeutics. They are also using their approach as a model to understand the circuitry of other disease-associated regions in the human genome.

Flipping a Genetic Switch on Obesity?

Illustration of a DNA switchWhen weight loss is the goal, the equation seems simple enough: consume fewer calories and burn more of them exercising. But for some people, losing and keeping off the weight is much more difficult for reasons that can include a genetic component. While there are rare genetic causes of extreme obesity, the strongest common genetic contributor discovered so far is a variant found in an intron of the FTO gene. Variations in this untranslated region of the gene have been tied to differences in body mass and a risk of obesity [1]. For the one in six people of European descent born with two copies of the risk variant, the consequence is carrying around an average of an extra 7 pounds [2].

Now, NIH-funded researchers reporting in The New England Journal of Medicine [3] have figured out how this gene influences body weight. The answer is not, as many had suspected, in regions of the brain that control appetite, but in the progenitor cells that produce white and beige fat. The researchers found that the risk variant is part of a larger genetic circuit that determines whether our bodies burn or store fat. This discovery may yield new approaches to intervene in obesity with treatments designed to change the way fat cells handle calories.

The team—led by Melina Claussnitzer of Beth Israel Deaconess Medical Center, Boston, and Manolis Kellis of the Massachusetts Institute of Technology (MIT), Cambridge—started with a basic question: where in the body does this variant act to influence weight? For the answer, the team turned to the NIH-funded Roadmap Epigenomics Project. There, they found comprehensive data on 127 human cell types and the occurrence of common chemical modifications that act like volume knobs to turn gene activity “up” or “down” based on changes in the way DNA is packaged. While the FTO gene is active in the human brain, the team couldn’t connect any differences there with obesity.

They began to wonder whether this obesity-risk variant affected FTO at all (and prior studies had suggested this [4]). Maybe it operated at a distance to change the expression of other protein-coding genes? Sure enough, further study in fat collected from patients showed that the obesity risk variant works in those progenitor cells to control the activity of two other genes, IRX3 andIRX5, both found quite a distance away.

The fat in people with the obesity risk variant and greater expression of IRX3 and IRX5 genes contains fewer beige cells than normal. Beige cells, which were discovered just three years ago [5], are produced sometimes by fat cell progenitors to burn rather than stockpile energy. This new evidence suggests that beige fat may play an unexpectedly important role in protecting against obesity.

Using a method they developed last year [6], the researchers traced the effects of the obesity risk variant to a single nucleotide change—a small typo in the DNA sequence that changes a “T” to a “C.” They then used the nifty CRISPR-Cas genome editing system (see Copy-Editing the Genome) to switch between this obesity risk variant and the protective variant in human cells. As the researchers did this, they saw fat cells turn energy-burning heat production off and back on again. In other words, the obesity signature in the cells could be turned on and off at the flip of this genetic switch!

They also showed in mice that the shift toward energy-burning beige cells led to weight loss. Animals engineered in a way that blocked Irx3 expression in adipose tissue became significantly thinner with no change in their eating or exercise habits. This new collection of evidence suggests that treatments designed to program fat cells to burn more energy (such as antagonists against the IRX3 or IRX5 proteins) might have similar benefits in people, and the researchers are working with collaborators in academia and industry to pursue this line of investigation.

This is a great example of how discoveries about genetic factors in common disease, uncovered by applying the genome-wide association study (GWAS) approach to large numbers of affected and unaffected individuals, are revealing critical and previously unknown pathways in human biology and medicine. This case also points out how our terminology may need attention, however; for the last several years, this genetic variant for obesity has been called “the FTO variant,” perhaps it should now be called “the IRX3/5 variant.”

Genes, of course, are only part of the story. It’s still important to eat healthy, limit your portions, and maintain a regular exercise program. Leading an active lifestyle both keeps weight down and improves the overall sense of well being.


[1] FTO genotype is associated with phenotypic variability of body mass index.Yang J, Loos RJ, Powell JE, TM, Frayling TM, Hirschhorn JN, Goddard ME, Visscher PM, et al. Nature. 2012 Oct 11;490(7419):267-72.

[2] A common variant in the FTO gene is associated with body mass index and predisposes to childhood and adult obesity. Frayling TM, Timpson NJ, Weedon MN, Morris AD, Smith GD, Hattersley AT, McCarthy MI, et al. Science. 2007 May 11;316(5826):889-94.

[3] FTO Obesity Variant Circuitry and Adipocyte Browning in Humans. Claussnitzer M, Dankel SN, Kim KH, Quon G, Meuleman W, Haugen C, Glunk V, Sousa IS, Beaudry JL, Puviindran V, Abdennur NA, Liu J, Svensson PA, Hsu YH, Drucker DJ, Mellgren G, Hui CC, Hauner H, Kellis M. N Engl J Med. 2015 Aug 19. [Epub ahead of print]

[4] Obesity-associated variants within FTO form long-range functional connections with IRX3. Smemo S, Tena JJ, Kim KH, Hui CC, Gomez-Skarmeta JL, Nobrega MA, et al. Nature 2014 Mar 20; 507(7492):371-375.

[5] Beige adipocytes are a distinct type of themogenic fat cell in mouse and human. Wu J, Boström P, Sparks LM, Schrauwen P, Spiegelman BM. Cell 2012 Jul 20:150(2):366-376.

[6] Leveraging cross-species transcription factor binding site patterns: from diabetes risk loci to disease mechanisms. Claussnitzer M, Dankel SN, Klocke Mellgren G, Hauner H, Laumen H, et al. Cell. 2014 Jan 16;156(1-2):343-58.


Manolis Kellis (Massachusetts Institute of Technology, Cambridge)

What are overweight and obesity? (National Heart, Lung, and Blood Institute/NIH)

NIH Roadmap Epigenomics Project

NIH Support: National Human Genome Research Institute; National Institute of General Medical Sciences

MiR-93 Controls Adiposity via Inhibition of Sirt7 and Tbx3

Impact Factor: 8.36 · DOI: 10.1016/j.celrep.2015.08.006

Conquering obesity has become a major socioeconomic challenge. Here, we show that reduced expression of the miR-25-93-106b cluster, or miR-93 alone, increases fat mass and, subsequently, insulin resistance. Mechanistically, we discovered an intricate interplay between enhanced adipocyte precursor turnover and increased adipogenesis. First, miR-93 controls Tbx3, thereby limiting self-renewal in early adipocyte precursors. Second, miR-93 inhibits the metabolic target Sirt7, which we identified as a major driver of in vivo adipogenesis via induction of differentiation and maturation of early adipocyte precursors. Using mouse parabiosis, obesity in mir-25-93-106b(-/-) mice could be rescued by restoring levels of circulating miRNA and subsequent inhibition of Tbx3 and Sirt7. Downregulation of miR-93 also occurred in obese ob/ob mice, and this phenocopy of mir-25-93-106b(-/-) was partially reversible with injection of miR-93 mimics. Our data establish miR-93 as a negative regulator of adipogenesis and a potential therapeutic option for obesity and the metabolic syndrome.

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Heroes in Basic Medical Research – Robert J. Lefkowitz

Author & Curator: Larry H Bernstein, MD, FCAP

Robert J. Lefkowitz, MD

Robert J. Lefkowitz MD, a Howard Hughes Medical Institute investigator who has spent his entire 39-year research career at the Duke University Medical Center, is sharing the 2012 Nobel Prize in Chemistry with Brian K. Kobilka of Stanford University School of Medicine, who was a post-doctoral fellow in Lefkowitz’s lab in the 1980s.

They are being recognized for their work on a class of cell surface receptors that have become the target of prescription drugs, including antihistamines, ulcer drugs and beta blockers to relieve hypertension, angina and coronary disease.

The receptors catch chemical signals from the outside and transmit their messages into the cell, providing the cell with information about changes occurring within the body. These particular receptors are called seven-transmembrane G protein-coupled receptors, or just “G-coupled receptors” for short. Serpentine in appearance, G-coupled receptors weave through the surface of the cell seven times.

The human genome contains code to make at least 1,000 different forms of these trans-membrane receptors, all of which are quite similar. The receptors also bear a strong resemblance to receptors that detect light in the eyes, smells in the nose and taste on the tongue. (See playlist of Lefkowitz science videos here.)

“Bob’s seminal discoveries related to G-protein coupled receptors ultimately became the basis for a great many medications that are in use today across many disease areas,” said Victor J. Dzau, MD, Chancellor for Health Affairs and CEO, Duke University Health System.  “He is an outstanding example of a physician-scientist whose impact can be seen in the lives of the countless patients who have benefited from his scientific discoveries. We are very proud of his magnificent achievements and grateful for his many contributions to Duke Medicine.”

After attending public elementary and junior high schools I entered The Bronx High School of Science (10th grade) in the autumn of 1956, graduating at age 16 in 1959. “Bronx Science” is one of several public high schools in New York City which admits students on the basis of a competitive examination. The student body, representing approximately the top 5% based on the exam, are gifted and interested in science and math. The accomplishments of graduates of this high school are quite remarkable. For example, I am the 8th Nobel Laureate to have graduated from this school, the 7 previous ones having received their prizes in Physics. For me, attending this school was a formative experience. Whereas in elementary and junior high school I was not greatly challenged, here I was among a group of remarkably bright, interesting and stimulating classmates. The curriculum featured many advanced classes at the college level. I was particularly drawn to chemistry and, as a result of taking these college level classes, I was able to receive full credit for two years of chemistry when I entered Columbia College in 1959. Thus I began as a college freshman with organic chemistry, a course generally taken by juniors.

The level of scholarship maintained by the student body was such that even with an average of about 94% my final class rank was about 100th out of 800. A classmate and friend at the time and at present, the famous geneticist David Botstein, had an almost identical average, a fact we tease each other about to this day.

Along with dozens of classmates, I moved on to Columbia University where I enrolled as a pre-medical student majoring in chemistry. The two year core curriculum in “Contemporary Civilization” was required of all students. With an emphasis on reading classic texts in history, philosophy, sociology and the political sciences and discussing these in small seminars, it was for me an opening to a whole new world. In addition, I took courses with and was exposed to, such intellectual giants as the literary critic Lionel Trilling, the cultural historian Jacques Barzun and the sociologist Daniel Bell, among others. I have very fond memories from this period of spending many hours in the public reading room at the 42nd Street New York Public Library, researching papers for those classes.

I also studied advanced Organic Chemistry with Cheves Walling and Physical Chemistry in a department which was strongly influenced by the then recently retired prominent physical organic chemist, Louis Hammett. However, the chemistry professor who had the most profound influence on me was actually a young Assistant Professor of Chemistry, Ronald Breslow. As a college senior I took an advanced seminar in biochemistry which he taught single handedly. This introduction to the chemistry of processes in living organisms really excited me in part, I suspect, because of his very lively teaching style. None of this, however, in any way diverted me from my goal of studying to become a practicing physician.

I greatly enjoyed my four years in medical school. I had dreamed about becoming a physician since grade school and now I was finally doing it. As a freshman immersed in the basic medical sciences I was able to deepen my interest in, and fascination with, biochemistry. Our biochemistry professors included a remarkable array of scholars (not that any of us appreciated that at the time). We heard lectures on metabolism from David Rittenberg, Chair of the Department; from David Shemin on porphyrins; from Irwin Chargaff on nucleic acids; and from David Nachmansohn on cholinergic neurotransmission.

One young professor left a lasting impression on me. Paul Marks was then a young academic hematologist who taught the Introduction to Clinical Medicine course in which we studied clinical problems for the first time, examined case histories, and looked at blood specimens. Not only was he a good clinician but he assigned readings from the basic science literature that were relevant in a very meaningful way to the cases we studied. This showed me how scientific information could be brought to bear on clinical problems. Among my classmates and friends in medical school was Harold Varmus, who was the co-recipient of the 1989 Nobel Prize for the discovery of oncogenes.

On July 1, 1968 I moved my family (now including the recently born Cheryl) to Rockville, Maryland to begin my research career at the NIH in nearby Bethesda, Maryland. I had been assigned, through a matching program, to work with Drs. Jesse Roth and Ira Pastan in the Clinical Endocrinology Branch of the National Institute of Arthritis and Metabolic Diseases (NIAMD), now known as NIDDK, the National Institute of Diabetes and Digestive and Kidney Diseases. I was a Clinical Associate, meaning that in addition to doing full time research ten months out of the year, for two months I also supervised a clinical endocrinology in-patient service. Because of this, I gained a remarkable exposure to unusual endocrine diseases which were under study at the time. An example of this was acromegaly.

It was the heyday of interest in second messenger signaling after the discovery of cAMP by Earl Sutherland. He would receive the Nobel Prize in Medicine and Physiology for this in 1971. One hormone after another was being shown to stimulate the enzyme adenylate cyclase thus increasing intracellular levels of cAMP. The idea that these different hormones might work through distinct receptors was talked about but was controversial. Moreover, at the time there were no direct methods for studying the receptors. I was assigned the challenging task of developing a radioligand binding method to study the putative receptors for adrenocorticotropic hormone (ACTH) in plasma membranes derived from an ACTH responsive adrenocortical carcinoma passaged in nude mice.

Recently, two Nobel Laureates, Mike Brown and Joe Goldstein, published a brief essay discussing the remarkable number of Nobel Laureates (9 so far) who have in common the fact that they came to the NIH as physicians during the brief space between 1964–1972 for postdoctoral research training. (1)

They dissect the unique convergence of circumstances which may have been responsible for this extraordinary result, including the quality of basic science mentors on the full time NIH staff, the competitiveness of “the best and the brightest” to obtain these positions during the Vietnam War years, and the now bygone emphasis on teaching of basic sciences in medical schools in the 1960s.

Lineages among Nobel Laureates are often commented upon. In my case, Jesse Roth had trained with Solomon Berson and Rosalyn Yalow whose development of radioimmunoassay led to the Nobel Prize in Medicine and Physiology to Yalow (1977) after Berson’s untimely death in 1972. Moreover, training in Ira Pastan’s laboratory contemporaneously with me was my medical school and house staff classmate and future Nobel Laureate, Harold Varmus. Ira had himself trained in the lab of another NIH career scientist, Earl Stadtman, who also trained a future Nobel Laureate, Mike Brown.

Dr. Edgar Haber, the Chief of Cardiology and a prominent immunochemist, allowed me to begin working in his lab. I was fascinated by receptors and what I saw as their potential to form the basis for a whole new field of research just waiting to be explored. I spent a great deal of time analyzing which receptor I should attempt to study. As an aspiring academic cardiologist I wanted to work on something related to the cardiovascular system. I also wanted a receptor known to be coupled to adenylate cyclase. I initially focused on two models, the cardiac glucagon and β-adrenergic receptors. However, my attention quickly became focused on the latter, for very practical reasons. Unlike the case for peptide hormones such as glucagon or ACTH, literally dozens, if not hundreds of analogs of adrenaline and noradrenaline, as well as their antagonists were available which could be chemically modified to develop the types of new tools which would need to be developed to study the receptors. These would include radioligands, photoaffinity probes, affinity chromatography matrices and the like. Moreover, the first β-adrenergic receptor blocker (“β-blocker”) had recently been approved for clinical use in the United States, adding further to the attractiveness of this target to me.

So in the early months of 1971 I began the quest to prove the existence of β-adrenergic receptors, to study their properties, to learn about their chemical nature, how they were regulated and how they functioned. This work has consumed me for the past forty years. Over the next several years in Boston, working mostly with membrane fractions derived from canine myocardium, I sought to develop radioligand binding approaches to tag the β-adrenergic receptors. I focused initially on the use of [3H]labeled catecholamines such as norepinephrine, which are agonists for the receptor. Specific saturable binding could be demonstrated, and I thought initially that we had developed a valid approach to label the receptors. However, it became increasingly clear over the next few years that the sites being labeled lacked many of the properties that would be expected for true physiological receptor binding sites. Coming to this realization was difficult.

During this time I also published some of the very first studies demonstrating GTP regulation of β-adrenergic receptor stimulated adenylate cyclase following after the work of Martin Rodbell on GTP regulation of glucagon sensitive adenylate cyclase. I was now a cardiology fellow. As at the NIH, nights on call were often spent in the lab doing experiments while hoping that my on call beeper would remain quiet. During these years, I had many stimulating and profitable discussions with Geoffrey Sharpe, a faculty member in the Nephrology Division with an interest in cell signaling and adenylate cyclase.

In work with postdoc Marc Caron in the spring of 1974, we succeeded in developing [3H]dihydroalprenolol. Contemporaneously, Gerald Aurbach at the NIH, and Alex Levitzki at the Hebrew University in Jerusalem also developed similar approaches using different radioligands. This was a watershed event because it finally opened the door to direct study of the receptors. Together with M.D./Ph.D. student Rusty Williams we developed comparable assays for the α-adrenergic receptors shortly thereafter.

Brian Kent Kobilka is an American physiologist and a corecipient of the 2012 Nobel Prize in Chemistry with Robert Lefkowitz for discoveries that reveal the inner workings of an important family G protein-coupled receptors.

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Metabolic Genomics and Pharmaceutics, Vol. 1 of BioMed Series D available on Amazon Kindle

Metabolic Genomics and Pharmaceutics, Vol. 1 of BioMed Series D available on Amazon Kindle

Reporter: Stephen S Williams, PhD


Leaders in Pharmaceutical Business Intelligence would like to announce the First volume of their BioMedical E-Book Series D:

Metabolic Genomics & Pharmaceutics, Vol. I

SACHS FLYER 2014 Metabolomics SeriesDindividualred-page2

which is now available on Amazon Kindle at

This e-Book is a comprehensive review of recent Original Research on  METABOLOMICS and related opportunities for Targeted Therapy written by Experts, Authors, Writers. This is the first volume of the Series D: e-Books on BioMedicine – Metabolomics, Immunology, Infectious Diseases.  It is written for comprehension at the third year medical student level, or as a reference for licensing board exams, but it is also written for the education of a first time baccalaureate degree reader in the biological sciences.  Hopefully, it can be read with great interest by the undergraduate student who is undecided in the choice of a career. The results of Original Research are gaining value added for the e-Reader by the Methodology of Curation. The e-Book’s articles have been published on the Open Access Online Scientific Journal, since April 2012.  All new articles on this subject, will continue to be incorporated, as published with periodical updates.

We invite e-Readers to write an Article Reviews on Amazon for this e-Book on Amazon.

All forthcoming BioMed e-Book Titles can be viewed at:

Leaders in Pharmaceutical Business Intelligence, launched in April 2012 an Open Access Online Scientific Journal is a scientific, medical and business multi expert authoring environment in several domains of  life sciences, pharmaceutical, healthcare & medicine industries. The venture operates as an online scientific intellectual exchange at their website and for curation and reporting on frontiers in biomedical, biological sciences, healthcare economics, pharmacology, pharmaceuticals & medicine. In addition the venture publishes a Medical E-book Series available on Amazon’s Kindle platform.

Analyzing and sharing the vast and rapidly expanding volume of scientific knowledge has never been so crucial to innovation in the medical field. WE are addressing need of overcoming this scientific information overload by:

  • delivering curation and summary interpretations of latest findings and innovations on an open-access, Web 2.0 platform with future goals of providing primarily concept-driven search in the near future
  • providing a social platform for scientists and clinicians to enter into discussion using social media
  • compiling recent discoveries and issues in yearly-updated Medical E-book Series on Amazon’s mobile Kindle platform

This curation offers better organization and visibility to the critical information useful for the next innovations in academic, clinical, and industrial research by providing these hybrid networks.

Table of Contents for Metabolic Genomics & Pharmaceutics, Vol. I

Chapter 1: Metabolic Pathways

Chapter 2: Lipid Metabolism

Chapter 3: Cell Signaling

Chapter 4: Protein Synthesis and Degradation

Chapter 5: Sub-cellular Structure

Chapter 6: Proteomics

Chapter 7: Metabolomics

Chapter 8:  Impairments in Pathological States: Endocrine Disorders; Stress

                   Hypermetabolism and Cancer

Chapter 9: Genomic Expression in Health and Disease 






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Sequencing yourself! and Learn more on Genome Sequencing on Tuesday, November 17, 2015 from 8am-5pm in the Joseph B. Martin Conference Center of the Harvard New Research Building at Harvard Medical School

Reporter: Aviva Lev-Ari, PhD, RN

Become one of the first humans to have your entire genome sequenced, while participating in an interactive set of presentations and debates about the promise and limitations of genome sequencing from some of the world’s leading genomic scientists.

The UYG Boston is an invitation-only, interactive symposium in which approximately 60 leaders from the Boston business and academic communities will have the opportunity to undergo whole genome sequencing, and to explore their own genome as part of an all-day educational conference with exciting presentations, debates and comments from some of the most thought-provoking leaders in the field of sequencing, informatics and genomic medicine.


  • Brigham Genome Medicine, Brigham and Women’s Hospital
  • Partners Personalized Medicine and Laboratory for Molecular Medicine
  • Precision Medicine Program at Brigham and Women’s Hospital
  • Department of Pathology at Brigham and Women’s Hospital
  • Analytic and Translational Genetics Unit, Massachusetts General Hospital
  • The Broad Institute of Harvard and MIT
  • Department of Pathology at Massachusetts General Hospital
  • Division of Genetics, Department of Medicine, Brigham and Women’s Hospital


Draft Agenda for UYG Agenda, November 17, 2015, NRB Rotunda Room

Registration is still open at this link:

Breakfast and Registration


Module 1: Understanding the Basics of Genetics and Genomics


Moderator: __________________________________

8:30 am -9:55 am

(10) Robert Green: Welcome and Introductory Remarks

(20) Stacey Gabriel: Technical Overview of Sequencing, Alignment and Variant Calling

(20) Heidi Rehm: Variant Classification and Lab Reporting: the Good, the Bad and the VUS

(20) Daniel MacArthur: Using Large Datasets to Explore Penetrance

(15) Questions and Discussion

Coffee Break

9:55 am – 10:10 am

Module 2: Sequencing and Informatics in Clinical Care


Moderator: __________________________________

10:10 am-11:30 noon

(20) Dick Maas: Sequencing in Undiagnosed Cases

(20) Kricket Seidman: Sequencing in the Care of Specific Diseases (Cardiomyopathy)

(20) Zak Kohane: Sequencing and Informatics

(20) Discussion

Luncheon: Understand Your Genome®

11:30 noon – 1:00 pm

Pechet Room

Lunch for those who have been sequenced or wish to learn to use the MyGenome web portal with the demo genome (seating is limited to 40 WGS attendees + 10 additional attendees):

(30) Erica Ramos: Clinical Whole Genome Sequencing in a Healthy Population

(30) Erica Ramos: MyGenome Web Portal Revealed

(30) Erica Ramos and Genetic Counselors: Holding and Exploring Your Own Genome

Lunch served separately for those who do not wish to explore MyGenome Web App

Module 3: Sequencing in Research: from Discovery to Patient Care


Moderator: __________________________________

1:00 pm – 2:40 pm

(10) Jeff Flier: Afternoon welcome and remarks

(20) Sek Kathiresan: Developing Medicines that Mimic Natural Genomic Successes

(20) Calum MacRae: Global Phenotyping and the Clinic of the Future

(10) Heidi Rehm: ClinGen and Matchmaker Exchange

(20) Robert Green: Clinical Outcomes Research in Sequencing

(20) Discussion

Module 4: Academic Medical Centers and Personalized/Precision Medicine


Moderator: __________________________________

2:40- 3:35

(15) Betsy Nabel: Direct-to Consumer Sequencing and the Academic Medical Center

(20) Jeff Golden: Precision Medicine, Regulation and Reimbursement

(20) Discussion

Afternoon Break


Module 5: Debate on the Benefits, Harms and Costs of Sequencing Health Individuals by Individual Speakers with the Entire Panel of Speakers and the Attendees


Five Minute Pro or Con by Each Speaker and Select Audience Members, Followed by Debate


“DNA Team” Captains: “There is Benefit” Jeff Golden/Jeff Flier

“RNA Team” Captains: “There is No Benefit” Sek Kathiresan/Betsy Nabel

Closing Remarks

4:55 – 5:00

(5) Robert Green

Wine and Cheese Reception for Speakers and Attendees



– See more at:


Register by July 15th to attend for $3,100!
After July 15th registration will be $3,500.

Pricing includes:

$2,900 TruGenome™ Predisposition Screen plus a conference registration fee


November 17, 2015
General Session: 8am – 5pm
Reception: 5pm – 7pm


The Joseph B. Martin Conference Center
Harvard Medical School
77 Avenue Louis Pasteur
Boston, MA 02115

Confirmed Speakers

  • George Church, PhD,
    Harvard Medical School
  • Stacey Gabriel, PhD
    The Broad Institute
  • Jeff Golden, MD
    Brigham and Women’s Hospital
  • Robert Green, MD, MPH
    Brigham and Women’s Hospital
  • Sek Kathiresan, MD
    Massachusetts General Hospital
  • Zak Kohane, MD, PhD
    Harvard Medical School
  • Richard Maas, MD, PhD
    Brigham and Women’s Hospital
  • Daniel MacArthur, PhD
    Massachusetts General Hospital
  • Calum MacRae, MD
    Brigham and Women’s Hospital
  • Betsy Nabel, MD
    Brigham and Women’s Hospital
  • Heidi Rehm, PhD
    Laboratory of Molecular Medicine
  • Christine Seidman, MD
    Brigham and Women’s Hospital


From: Robert Green <>

Date: Tuesday, July 7, 2015 at 1:46 PM

Subject: Learn about genome sequencing by sequencing yourself!

Robert C. Green, MD, MPH

Director, G2P Research Program

Associate Director for Research, Partners Center for Personalized Genetic Medicine

Division of Genetics, Department of Medicine

Brigham and Women’s Hospital and Harvard Medical School

EC Alumnae Building, Suite 301, 41 Avenue Louis Pasteur, Boston, MA 02115                                    

(office) 617-264-5834, (fax) 617-264-3018, (cell) 617-966-3216



Dear Colleagues:

We are inviting you, as one of a small group of forward looking thought leaders, to attend an exciting educational and experiential event: the Boston “Understand your Genome” conference. This conference will take place the day before this year’s Partners Personalized Medicine Conference at Harvard Medical School and will have two components.

First, a panel of world-renowned speakers will discuss the current progress and promise of genomic medicine, and debate the controversial issues surrounding the sequencing of healthy individuals for prediction and prevention.

Second, the conference will provide you with the option to become one of the first people on the planet to have your whole genome sequenced at a CLIA facility where a report will be generated by a board certified molecular geneticist on 1,691 genes with well-established associations to 1,232 Mendelian conditions, and 11 genes associated with responses to 16 different medications.

This conference is a non-profit educational event that is sponsored by the Division of Genetics, in the Department of Medicine at Brigham and Women’s Hospital with co-sponsorship by Partners Personalized Medicine and the Laboratory for Molecular Medicine, the Precision Medicine Program at Brigham and Women’s Hospital, the Department of Pathology at Brigham and Women’s Hospital, the Analytic and Translational Genetics Unit at Massachusetts General Hospital, the Department of Pathology at Massachusetts General Hospital and the Broad Institute. Together, we have assembled a remarkable panel of speakers for the first component of the program.

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