Robert A. Kesterson

Targeted disruption of the melanocortin-4 receptor results in obesity in mice

The melanocortin-4 receptor (MC4-R) is a G protein-coupled, seven-transmembrane receptor expressed in the brain. Inactivation of this receptor by gene targeting results in mice that develop a maturity onset obesity syndrome associated with hyperphagia, hyperinsulinemia, and hyperglycemia. This syndrome recapitulates several of the characteristic features of the agouti obesity syndrome, which results from ectopic expression of agouti protein, a pigmentation factor normally expressed in the skin. Our data identify a novel signaling pathway in the mouse for body weight regulation and support a model in which the primary mechanism by which agouti induces obesity is chronic antagonism of the MC4-R.

Abnormal adaptations to stress and impaired cardiovascular function in mice lacking corticotropin-releasing hormone receptor-2

The actions of corticotropin-releasing hormone (Crh), a mediator of endocrine and behavioural responses to stress, and the related hormone urocortin (Ucn) are coordinated by two receptors, Crhr1 (encoded by Crhr) and Crhr2 (refs 4,5). These receptors may exhibit distinct functions due to unique tissue distribution and pharmacology. Crhr-null mice have defined central functions for Crhr1 in anxiety and neuroendocrine stress responses. Here we generate Crhr2−/− mice and show that Crhr2 supplies regulatory features to the hypothalamic-pituitary-adrenal axis (HPA) stress response. Although initiation of the stress response appears to be normal, Crhr2−/− mice show early termination of adrenocorticotropic hormone (Acth) release, suggesting that Crhr2 is involved in maintaining HPA drive. Crhr2 also appears to modify the recovery phase of the HPA response, as corticosterone levels remain elevated 90 minutes after stress in Crhr2−/− mice. In addition, stress-coping behaviours associated with dearousal are reduced in Crhr2–/– mice. We also demonstrate that Crhr2 is essential for sustained feeding suppression (hypophagia) induced by Ucn. Feeding is initially suppressed in Crhr2−/− mice following Ucn, but Crhr2−/− mice recover more rapidly and completely than do wild-type mice. In addition to central nervous system effects, we found that, in contrast to wild-type mice, Crhr2−/− mice fail to show the enhanced cardiac performance or reduced blood pressure associated with systemic Ucn, suggesting that Crhr2 mediates these peripheral haemodynamic effects. Moreover, Crhr2−/− mice have elevated basal blood pressure, demonstrating that Crhr2 participates in cardiovascular homeostasis. Our results identify specific responses in the brain and periphery that involve Crhr2.

Deletion of PPARγ in adipose tissues of mice protects against high fat diet-induced obesity and insulin resistance

Peroxisome proliferator-activated receptor γ (PPARγ) plays a crucial role in adipocyte differentiation, glucose metabolism, and other physiological processes. To further explore the role of PPARγ in adipose tissues, we used a Cre/loxP strategy to generate adipose-specific PPARγ knockout mice. These animals exhibited marked abnormalities in the formation and function of both brown and white adipose tissues. When fed a high-fat diet, adipose-specific PPARγ knockout mice displayed diminished weight gain despite hyperphagia, had diminished serum concentrations of both leptin and adiponectin, and did not develop glucose intolerance or insulin resistance. Characterization of in vivo glucose dynamics pointed to improved hepatic glucose metabolism as the basis for preventing high-fat diet-induced insulin resistance. Our findings further illustrate the essential role for PPARγ in the development of adipose tissues and suggest that a compensatory induction of hepatic PPARγ may stimulate an increase in glucose disposal by the liver.

Disruption of Intraflagellar Transport in Adult Mice Leads to Obesity and Slow-Onset Cystic Kidney Disease

The assembly of primary cilia is dependent on intraflagellar transport (IFT), which mediates the bidirectional movement of proteins between the base and tip of the cilium. In mice, congenic mutations disrupting genes required for IFT (e.g., Tg737 or the IFT kinesin Kif3a) are embryonic lethal, whereas kidney-specific disruption of IFT results in severe, rapidly progressing cystic pathology. Although the function of primary cilia in most tissues is unknown, in the kidney they are mechanosenstive organelles that detect fluid flow through the tubule lumen. The loss of this flow-induced signaling pathway is thought to be a major contributing factor to cyst formation. Recent data also suggest that there is a connection between ciliary dysfunction and obesity as evidenced by the discovery that proteins associated with human obesity syndromes such as Alström and Bardet-Biedl localize to this organelle. To more directly assess the importance of cilia in postnatal life, we utilized conditional alleles of two ciliogenic genes (Tg737 and Kif3a) to systemically induce cilia loss in adults. Surprisingly, the cystic kidney pathology in these mutants is dependent on the time at which cilia loss was induced, suggesting that cyst formation is not simply caused by impaired mechanosensation. In addition to the cystic pathology, the conditional cilia mutant mice become obese, are hyperphagic, and have elevated levels of serum insulin, glucose, and leptin. We further defined where in the body cilia are required for normal energy homeostasis by disrupting cilia on neurons throughout the central nervous system and on pro-opiomelanocortin-expressing cells in the hypothalamus, both of which resulted in obesity. These data establish that neuronal cilia function in a pathway regulating satiety responses.

Identification of Adropin as a Secreted Factor Linking Dietary Macronutrient Intake with Energy Homeostasis and Lipid Metabolism

Obesity and nutrient homeostasis are linked by mechanisms that are not fully elucidated. Here we describe a secreted protein, adropin, encoded by a gene, Energy Homeostasis Associated (Enho), expressed in liver and brain. Liver Enho expression is regulated by nutrition: lean C57BL/6J mice fed high-fat diet (HFD) exhibited a rapid increase, while fasting reduced expression compared to controls. However, liver Enho expression declines with diet-induced obesity (DIO) associated with 3 months of HFD or with genetically induced obesity, suggesting an association with metabolic disorders in the obese state. In DIO mice, transgenic overexpression or systemic adropin treatment attenuated hepatosteatosis and insulin resistance independently of effects on adiposity or food intake. Adropin regulated expression of hepatic lipogenic genes and adipose tissue peroxisome proliferator-activated receptor gamma, a major regulator of lipogenesis. Adropin may therefore be a factor governing glucose and lipid homeostasis, which protects against hepatosteatosis and hyperinsulinemia associated with obesity.

Mice with altered serotonin 2C receptor RNA editing display characteristics of Prader-Willi Syndrome

RNA transcripts encoding the 2C-subtype of serotonin (5HT(2C)) receptor undergo up to five adenosine-to-inosine editing events to encode twenty-four protein isoforms. To examine the effects of altered 5HT(2C) editing in vivo, we generated mutant mice solely expressing the fully-edited (VGV) isoform of the receptor. Mutant animals present phenotypic characteristics of Prader-Willi syndrome (PWS) including a failure to thrive, decreased somatic growth, neonatal muscular hypotonia, and reduced food consumption followed by post-weaning hyperphagia. Though previous studies have identified alterations in both 5HT(2C) receptor expression and 5HT(2C)-mediated behaviors in both PWS patients and mouse models of this disorder, to our knowledge the 5HT(2C) gene is the first locus outside the PWS imprinted region in which mutations can phenocopy numerous aspects of this syndrome. These results not only strengthen the link between the molecular etiology of PWS and altered 5HT(2C) expression, but also demonstrate the importance of normal patterns of 5HT(2C) RNA editing in vivo.

Analysis of osteocalcin expression in transgenic mice reveals a species difference in vitamin D regulation of mouse and human osteocalcin genes

A line of transgenic mice expressing a human osteocalcin genomic fragment (hOClocus) and a murine MC3T3‐E1 cell line containing a stably integrated human osteocalcin promoter construct have been developed to characterize the osteogenic and hormonal regulation of human osteocalcin in vivo and in vitro. In this study, we used these models to demonstrate a species difference in the regulation of the mouse and human osteocalcin genes by vitamin D. Repeated administration of 1,25‐dihydroxyvitamin D3 (1,25(OH)2D3) to mice carrying the hOClocus transgene resulted in striking increases in serum human osteocalcin, whereas serum mouse osteocalcin levels were unchanged after 24 h and only modestly increased 48 h after the second dose of hormone. 1,25(OH)2D3 increased human calvarial mRNA expression by 1.8‐fold and slightly decreased mouse osteocalcin mRNA levels by approximately 1.2‐fold. Furthermore, treatment of primary calvarial osteoblasts from these mice with 1,25(OH)2D3 increased human osteocalcin production but inhibited mouse osteocalcin protein accumulation. To investigate further the mechanism for the apparent species difference in vitamin D3 induction of mouse and human osteocalcin, we examined the effect of 1,25(OH)2D3 in an MC3T3‐E1 cell line (MC4) containing a stably integrated 3900 bp osteocalcin promoter‐luciferase construct. Treatment of MC4 cells with ascorbic acid resulted in parallel increases of the endogenous mouse osteocalcin protein and luciferase reporter activity over a 12‐day period. Continuous exposure of MC4 cells to 1,25(OH)2D3 resulted in time‐and dose‐dependent increases in the activity of the phOC3900 luciferase construct. By contrast, the hormone had no effect on mouse osteocalcin protein concentrations and inhibited its induction by ascorbic acid. However, when cells were treated acutely with 1,25(OH)2D3 at later times during growth in ascorbic acid, the induction of mouse osteocalcin protein was only partially inhibited. In conclusion, our results indicate that common osteogenic signals regulate both mouse and human osteocalcin gene expression, but the mouse gene is resistant to induction by vitamin D. This species difference in vitamin D regulation of osteocalcin appears to result from the failure of 1,25(OH)2D3 to transcriptionally activate the mouse osteocalcin gene.

Leptin resistance is a secondary consequence of the obesity in ciliopathy mutant mice

Although primary cilia are well established as important sensory and signaling structures, their function in most tissues remains unknown. Obesity is a feature associated with some syndromes of cilia dysfunction, such as Bardet-Biedl syndrome (BBS) and Alström syndrome, as well as in several cilia mutant mouse models. Recent data indicate that obesity in BBS mutant mice is due to defects in leptin receptor trafficking and leptin resistance. Furthermore, induction of cilia loss in leptin-responsive proopiomelanocortin neurons results in obesity, implicating cilia on hypothalamic neurons in regulating feeding behavior. Here, we directly test the importance of the cilium as a mediator of the leptin response. In contrast to the current dogma, a longitudinal study of conditional Ift88 cilia mutant mice under different states of adiposity indicates that leptin resistance is present only when mutants are obese. Our studies show that caloric restriction leads to an altered anticipatory feeding behavior that temporarily abrogates the anorectic actions of leptin despite normalized circulating leptin levels. Interestingly, preobese Bbs4 mutant mice responded to the anorectic effects of leptin and did not display other phenotypes associated with defective leptin signaling. Furthermore, thermoregulation and activity measurements in cilia mutant mice are inconsistent with phenotypes previously observed in leptin deficient ob/ob mice. Collectively, these data indicate that cilia are not directly involved in leptin responses and that a defect in the leptin signaling axis is not the initiating event leading to hyperphagia and obesity associated with cilia dysfunction.

ST6Gal-I Regulates Macrophage Apoptosis via α2-6 Sialylation of the TNFR1 Death Receptor

Background: The functional relevance of ST6Gal-I down-regulation during monocyte activation/macrophage differentiation is not well understood. Results: Cell and transgenic mouse models suggest that ST6Gal-I-mediated sialylation of TNFR1 blocks TNFα-induced apoptosis. Conclusion: ST6Gal-I down-regulation may limit monocyte/macrophage lifespan by sensitizing cells to apoptosis via TNFR1 hyposialylation. Significance: This is the first determination that TNFR1 function is regulated by its glycan structure. Macrophages play a central role in innate immunity, however mechanisms regulating macrophage survival are not fully understood. Herein we describe a novel apoptotic pathway involving α2-6 sialylation of the TNFR1 death receptor by the ST6Gal-I sialyltransferase. Variant glycosylation of TNFR1 has not previously been implicated in TNFR1 function, and little is known regarding the TNFR1 glycan composition. To study sialylation in macrophages, we treated U937 monocytic cells with PMA, which stimulates both macrophage differentiation and apoptosis. Interestingly, macrophage differentiation induces ST6Gal-I down-regulation, leading to reduced α2-6 sialylation of selected receptors. To prevent loss of α2-6 sialylation, we forced constitutive expression of ST6Gal-I, and found that this strongly inhibited PMA-induced apoptosis. Given that PMA-mediated apoptosis is thought to result from up-regulation of TNFα, which then activates TNFR1, we next evaluated the α2-6 sialylation of TNFR1. U937 cells with forced ST6Gal-I displayed TNFR1 with elevated α2-6 sialylation, and this was associated with diminished TNFα-stimulated apoptosis. Correspondingly, removal of α2-6 sialylation from TNFR1 through either neuraminidase treatment or expression of ST6Gal-I shRNA markedly enhanced TNFα-mediated apoptosis. To confirm the physiologic importance of TNFR1 sialylation, we generated overexpressing ST6Gal-I transgenic mice. Peritoneal macrophages from transgenic lines displayed TNFR1 with elevated α2-6 sialylation, and these cells were significantly protected against TNFα-stimulated apoptosis. Moreover, greater numbers of thioglycollate-induced peritoneal cells were observed in transgenic mice. These collective results highlight a new mechanism of TNFR1 regulation, and further intimate that loss of α2-6 sialylation during macrophage differentiation may limit macrophage lifespan by sensitizing cells to TNFα-stimulated apoptosis.

Hyperphagia-mediated obesity in transgenic mice misexpressing the RNA editing enzyme ADAR2

ADAR2 is a double-stranded RNA-specific adenosine deaminase involved in the editing of mammalian RNAs by the site-specific conversion of adenosine to inosine. To examine the physiologic consequences resulting from ADAR2 misexpression, we have generated mutant mice expressing either wild-type or deaminase-deficient ADAR2 transgenes under the control of the human cytomegalovirus promoter. Transgenic mice expressing either wild-type or inactive ADAR2 isoforms demonstrated adult onset obesity characterized by hyperglycemia, hyperleptinemia, and increased adiposity. Paired feeding analysis revealed that mutant mice on caloric restriction had a growth rate and body composition indistinguishable from wild-type littermates, indicating that the observed obesity predominantly results from hyperphagia rather than a metabolic derangement. The observation that expression of catalytically inactive ADAR2 also is capable of producing an obese phenotype in mutant animals suggests that ADAR2 may possess additional biological activities beyond those required for the site-selective deamination of adenosine or may interfere with the actions of other double-stranded RNA-specific binding proteins in the cell.