Regulator of Calcineurin 1 helps coordinate whole-body metabolism and thermogenesis.

Increasing non-shivering thermogenesis (NST), which expends calories as heat rather than storing them as fat, is championed as an effective way to combat obesity and metabolic disease. Innate mechanisms constraining the capacity for NST present a fundamental limitation to this approach, yet are not well understood. Here, we provide evidence that Regulator of Calcineurin 1 (RCAN1), a feedback inhibitor of the calcium-activated protein phosphatase calcineurin (CN), acts to suppress two distinctly different mechanisms of non-shivering thermogenesis (NST): one involving the activation of UCP1 expression in white adipose tissue, the other mediated by sarcolipin (SLN) in skeletal muscle. UCP1 generates heat at the expense of reducing ATP production, whereas SLN increases ATP consumption to generate heat. Gene expression profiles demonstrate a high correlation between Rcan1 expression and metabolic syndrome. On an evolutionary timescale, in the context of limited food resources, systemic suppression of prolonged NST by RCAN1 might have been beneficial; however, in the face of caloric abundance, RCAN1-mediated suppression of these adaptive avenues of energy expenditure may now contribute to the growing epidemic of obesity.

Regulator of calcineurin 1 modulates expression of innate anxiety and anxiogenic responses to selective serotonin reuptake inhibitor treatment.

Regulator of calcineurin 1 (RCAN1) controls the activity of calcium/calmodulin-dependent phosphatase calcineurin (CaN), which has been implicated in human anxiety disorders. Previously, we reported that RCAN1 functioned as an inhibitor of CaN activity in the brain. However, we now find enhanced phosphorylation of a CaN substrate, cAMP response element-binding protein (CREB), in the brains of Rcan1 knock-out (KO) mice. Consistent with enhanced CREB activation, we also observe enhanced expression of a CREB transcriptional target, brain-derived neurotrophic factor (BDNF) in Rcan1 KO mice. We also discovered that RCAN1 deletion or blockade of RCAN1-CaN interaction reduced CaN and protein phosphatase-1 localization to nuclear-enriched protein fractions and promoted CREB activation. Because of the potential links between CREB, BDNF, and anxiety, we examined the role of RCAN1 in the expression of innate anxiety. Rcan1 KO mice displayed reduced anxiety in several tests of unconditioned anxiety. Acute pharmacological inhibition of CaN rescued these deficits while transgenic overexpression of human RCAN1 increased anxiety. Finally, we found that Rcan1 KO mice lacked the early anxiogenic response to the selective serotonin reuptake inhibitor (SSRI) fluoxetine and had improved latency for its therapeutic anxiolytic effects. Together, our study suggests that RCAN1 plays an important role in the expression of anxiety-related and SSRI-related behaviors through CaN-dependent signaling pathways. These results identify RCAN1 as a mediator of innate emotional states and possible therapeutic target for anxiety.

Contribution of hexose-6-phosphate dehydrogenase to NADPH content and redox environment in the endoplasmic reticulum.

BACKGROUND: Hexose-6-phosphate dehydrogenase (H6PD) has been considered to be a main source of NADPH in the endoplasmic reticulum. It provides reducing equivalents to 11-hydroxysteroid dehydrogenase type 1 for in situ re-activation of glucocorticoids. H6PD null mice indeed show signs of glucocorticoid deficiency, but also suffer from a skeletal myopathy mainly affecting fast twitch muscles, in which the unfolded protein response (UPR) is activated. Thus, H6PD may have additional functions in muscle. MATERIALS AND METHODS: To determine the contribution of H6PD to total microsomal NADPH content, we measured NADPH in microsomes from liver and quadriceps, gastrocnemius and soleus muscles. To evaluate the effect of H6PD deficiency on microsomal thiol-disulfide redox environment, we measured reduced and oxidized glutathione and free protein thiols. RESULTS AND CONCLUSIONS: H6PD deficiency decreased but did not eliminate NADPH content in liver and soleus microsomes. Thus there must be other sources of NADPH within the endoplasmic/sarcoplasmic reticulum. Levels of reduced glutathione and free protein thiols were decreased in gastrocnemius muscle from null mice, indicating a more oxidative environment. Such alterations in redox environment may underlie the myopathy and UPR activation in H6PD null mice. GENERAL SIGNIFICANCE: H6PD plays a role in maintaining normal NADPH levels and redox environment inside the endoplasmic reticulum. Intrinsic differences in ER metabolism may explain the differing effects of H6PD deficiency in different tissues.

Ablation of whirlin long isoform disrupts the USH2 protein complex and causes vision and hearing loss.

Mutations in whirlin cause either Usher syndrome type II (USH2), a deafness-blindness disorder, or nonsyndromic deafness. The molecular basis for the variable disease expression is unknown. We show here that only the whirlin long isoform, distinct from a short isoform by virtue of having two N-terminal PDZ domains, is expressed in the retina. Both long and short isoforms are expressed in the inner ear. The N-terminal PDZ domains of the long whirlin isoform mediates the formation of a multi-protein complex that includes usherin and VLGR1, both of which are also implicated in USH2. We localized this USH2 protein complex to the periciliary membrane complex (PMC) in mouse photoreceptors that appears analogous to the frog periciliary ridge complex. The latter is proposed to play a role in photoreceptor protein trafficking through the connecting cilium. Mice carrying a targeted disruption near the N-terminus of whirlin manifest retinal and inner ear defects, reproducing the clinical features of human USH2 disease. This is in contrast to mice with mutations affecting the C-terminal portion of whirlin in which the phenotype is restricted to the inner ear. In mice lacking any one of the USH2 proteins, the normal localization of all USH2 proteins is disrupted, and there is evidence of protein destabilization. Taken together, our findings provide new insights into the pathogenic mechanism of Usher syndrome. First, the three USH2 proteins exist as an obligatory functional complex in vivo, and loss of one USH2 protein is functionally close to loss of all three. Second, defects in the three USH2 proteins share a common pathogenic process, i.e., disruption of the PMC. Third, whirlin mutations that ablate the N-terminal PDZ domains lead to Usher syndrome, but non-syndromic hearing loss will result if they are spared.

Studies on the very large G protein-coupled receptor: from initial discovery to determining its role in sensorineural deafness in higher animals.

The very large G protein-coupled receptor 1 (VLGRI), also known as MASS1 or GPR98, is most notable among the family of adhesion-GPCR for its size. Encoded by an 18.9 kb open reading frame, the approximately 700 kDa primary translation product is by far the largest GPCR and additionally, the largest cell surface protein known to date. The large ectodomain of the protein contains several repeated motifs, including some 35 calcium binding, Calx-beta repeats and seven copies of an epitempin repeat thought to be associated with the development of epilepsy. The extreme carboxy-terminus contains a consensus PDZ ligand sequence, suggesting interactions with other cytosolic or cytoskeletal proteins. At least two spontaneous and two targeted mutant mouse lines are currently known. The mutant mice present with sensitivity to audiogenic seizures but also have cochlear defects and significant, progressive hearing impairment. Although its ligand is currently unknown, VLGR1 is one of the few adhesion-GPCR family members in which mutations have been shown to be responsible for a human malady. Mutations in VLGRI in humans result in one form (2C) of Usher syndrome, the most common genetic cause of combined blindness and deafness.

Physiological roles of 11 beta-hydroxysteroid dehydrogenase type 1 and hexose-6-phosphate dehydrogenase.

PURPOSE OF REVIEW: Inactive cortisone is converted to active cortisol by the reductase activity of 11 beta-hydroxysteroid dehydrogenase type 1, which can thus increase glucocorticoid effects in target tissues. This paper reviews the functional role(s) of 11 beta-hydroxysteroid dehydrogenase type 1 and examines factors influencing its activity. RECENT FINDINGS: In obese humans, 11 beta-hydroxysteroid dehydrogenase type 1 is relatively highly expressed in adipose tissue. In mice, overexpression of 11 beta-hydroxysteroid dehydrogenase type 1 in adipose or liver causes obesity or insulin resistance, respectively, whereas mice lacking 11 beta-hydroxysteroid dehydrogenase type 1 resist diet-induced obesity and are insulin-sensitive. Thus, 11 beta-hydroxysteroid dehydrogenase type 1 is a promising drug target for treating the metabolic syndrome and type 2 diabetes. Studies in vitro and in mutant mice demonstrate that the reductase activity of 11 beta-hydroxysteroid dehydrogenase type 1 depends on reduced nicotinamide adenine dinucleotide phosphate synthesized within the endoplasmic reticulum by hexose-6-phosphate dehydrogenase. Apparent cortisone reductase deficiency is characterized by androgen excess in women or children and decreased urinary excretion of cortisol metabolites. Although polymorphisms in the genes encoding 11 beta-hydroxysteroid dehydrogenase type 1 and hexose-6-phosphate dehydrogenase were initially implicated in this condition, subsequent reports have not confirmed this. SUMMARY: Hexose-6-phosphate dehydrogenase and 11 beta-hydroxysteroid dehydrogenase type 1 may play important roles in the pathogenesis of obesity and metabolic syndrome. Although the importance of polymorphisms in the corresponding genes remains uncertain, rare mutations have not been ruled out.

Abnormalities of glucose homeostasis and the hypothalamic-pituitary-adrenal axis in mice lacking hexose-6-phosphate dehydrogenase.

Hexose-6-phosphate dehydrogenase (EC 1.1.1.47) catalyzes the conversion of glucose 6-phosphate to 6-phosphogluconolactone within the lumen of the endoplasmic reticulum, thereby generating reduced nicotinamide adenine dinucleotide phosphate. Reduced nicotinamide adenine dinucleotide phosphate is a necessary cofactor for the reductase activity of 11beta-hydroxysteroid dehydrogenase type 1 (EC 1.1.1.146), which converts hormonally inactive cortisone to active cortisol (in rodents, 11-dehydrocorticosterone to corticosterone). Mice with targeted inactivation of hexose-6-phosphate dehydrogenase lack 11beta-hydroxysteroid dehydrogenase type 1 reductase activity, whereas dehydrogenase activity (corticosterone to 11-dehydrocorticosterone) is increased. We now report that both glucose output and glucose use are abnormal in these mice. Mutant mice have fasting hypoglycemia. In mutant primary hepatocytes, glucose output does not increase normally in response to glucagon. Mutant animals have lower hepatic glycogen content when fed and cannot mobilize it normally when fasting. As assessed by RT-PCR, responses of hepatic enzymes to fasting are blunted; enzymes involved in gluconeogenesis (phosphoenolpyruvate carboxykinase, tyrosine aminotransferase) are not appropriately up-regulated, and expression of glucokinase, an enzyme required for glycolysis, is not suppressed. Corticosterone has attenuated effects on expression of these enzymes in cultured mutant primary hepatocytes. Mutant mice have increased sensitivity to insulin, as assessed by homeostatic model assessment values and by increased glucose uptake by the muscle. The hypothalamic-pituitary-adrenal axis is also abnormal. Circulating ACTH, deoxycorticosterone, and corticosterone levels are increased in mutant animals, suggesting decreased negative feedback on the hypothalamic-pituitary-adrenal axis. Comparison with other animal models of adrenal insufficiency suggests that many of the observed abnormalities can be explained by blunted intracellular corticosterone actions, despite elevated circulating levels of this hormone.

Hexose 6-phosphate dehydrogenase (H6PD) and corticosteroid metabolism.

Cortisone or (in rodents) 11-dehydrocorticosterone are reduced to cortisol or corticosterone, respectively, by the oxo-reductase activity of 11beta-hydroxysteroid dehydrogenase type 1 (11-HSD1). This requires NADPH, generated by hexose-6-phosphate dehydrogenase (H6PD), a component of the pentose phosphate pathway. H6PD is located along with 11-HSD1 in the lumen of the endoplasmic reticulum (ER). Increasing or decreasing expression levels of H6PD in cultured cells has corresponding effects on the reductase activity of 11-HSD1. Mice carrying a targeted mutation in H6PD have drastically decreased 11-HSD1 oxo-reductase activity, but their 11-dehydrogenase activity is increased. They have many phenotypic features in common with mice carrying a mutation of 11-HSD1 itself. Polymorphisms in both H6PD and 11-HSD1 were originally identified in patients with apparent cortisone reductase deficiency (who have signs of hyperandrogenism and decreased urinary excretion of cortisol versus cortisone metabolites). However, these polymorphisms do not have detectable biochemical or physiologic effects when prospectively ascertained.

The very large G-protein-coupled receptor VLGR1: a component of the ankle link complex required for the normal development of auditory hair bundles.

Sensory hair bundles in the inner ear are composed of stereocilia that can be interconnected by a variety of different link types, including tip links, horizontal top connectors, shaft connectors, and ankle links. The ankle link antigen is an epitope specifically associated with ankle links and the calycal processes of photoreceptors in chicks. Mass spectrometry and immunoblotting were used to identify this antigen as the avian ortholog of the very large G-protein-coupled receptor VLGR1, the product of the Usher syndrome USH2C (Mass1) locus. Like ankle links, Vlgr1 is expressed transiently around the base of developing hair bundles in mice. Ankle links fail to form in the cochleae of mice carrying a targeted mutation in Vlgr1 (Vlgr1/del7TM), and the bundles become disorganized just after birth. FM1-43 [N-(3-triethylammonium)propyl)-4-(4-(dibutylamino)styryl) pyridinium dibromide] dye loading and whole-cell recordings indicate mechanotransduction is impaired in cochlear, but not vestibular, hair cells of early postnatal Vlgr1/del7TM mutant mice. Auditory brainstem recordings and distortion product measurements indicate that these mice are severely deaf by the third week of life. Hair cells from the basal half of the cochlea are lost in 2-month-old Vlgr1/del7TM mice, and retinal function is mildly abnormal in aged mutants. Our results indicate that Vlgr1 is required for formation of the ankle link complex and the normal development of cochlear hair bundles.

Analysis of the very large G-protein coupled receptor gene (Vlgr1/Mass1/USH2C) in zebrafish.

Very Large G-protein coupled Receptor-1 (VLGR1/Mass1/USH2C) is the largest known cell surface protein in vertebrates. Mutations in VLGR1 are associated with audiogenic epilepsy in mice and Usher syndrome (sensorineural deafness and retinitis pigmentosa) in humans. We characterized the zebrafish VLGR1 gene (vlgr1). It is 51% identical to human VLGR1 in amino acid sequence, but is 64% identical in the 7-transmembrane and cytoplasmic domains. It is 6199 amino acids in size and is encoded by a 19.2 kb mRNA. All introns correspond in location and phase to those of the human and mouse genes. In situ hybridization studies of zebrafish embryos demonstrate vlgr1 expression in the developing central nervous system, particularly in the hypothalamus, epiphysis and in the rhombic lips. Expression in the eye is associated with the optic nerve. Further studies using zebrafish may help ascertain the role of Vlgr1 in neural development.

Loss of the transmembrane and cytoplasmic domains of the very large G-protein-coupled receptor-1 (VLGR1 or Mass1) causes audiogenic seizures in mice.

At approximately 6300 amino acids, very large G-protein-coupled receptor-1 (VLGR1, also termed Mass1) is the largest known cell surface protein. It is expressed at high levels within the embryonic nervous system, especially the ventricular zone. A naturally occurring nonsense mutation in VLGR1, V2250X, is linked with susceptibility to audiogenic seizures in mice. Interpretation of this finding is complicated by the existence of splice and transcriptional variants. We targeted the transmembrane and cytoplasmic domains of VLGR1, yielding a gene encoding the complete ectodomain of VLGR1 fused to antigenic tags (VLGR/del7TM). Homozygous mutant mice are susceptible to audiogenic seizures. Western blots detect a single very high molecular weight protein in brain extracts from VLGR/del7TM mice. These findings suggest that loss of VLGR1 transmembrane and cytoplasmic domains underlies the seizure phenotype in both mutant mouse strains, perhaps by disrupting signals regulating neural development.

Heat shock transcription factor 2 is not essential for embryonic development, fertility, or adult cognitive and psychomotor function in mice.

Members of the heat shock factor (HSF) family are evolutionarily conserved regulators that share a highly homologous DNA-binding domain. In mammals, HSF1 is the main factor controlling the stress-inducible expression of Hsp genes while the functions of HSF2 and HSF4 are less clear. Based on its developmental profile of expression, it was hypothesized that HSF2 may play an essential role in brain and heart development, spermatogenesis, and erythroid differentiation. To directly assess this hypothesis and better understand the underlying mechanisms that require HSF2, we generated Hsf2 knockout mice. Here, we report that Hsf2(-/-) mice are viable and fertile and exhibit normal life span and behavioral functions. We conclude that HSF2, most probably because its physiological roles are integrated into a redundant network of gene regulation and function, is dispensable for normal development, fertility, and postnatal psychomotor function.

Very large G protein-coupled receptor-1, the largest known cell surface protein, is highly expressed in the developing central nervous system.

We previously identified a member of the G protein-coupled receptor family, very large G protein-coupled receptor-1 (VLGR1). VLGR1 has a large ectodomain containing multiple calcium exchanger beta repeats that resemble regulatory domains of sodium-calcium exchanger proteins. Similar repeats are found in the extracellular aggregation factor of marine sponges, which mediates species-specific cell aggregation. We now report that the protein encoded by the originally described human cDNA (now termed VLGR1a) is, in fact, at 1967 amino acids, the smallest of three expressed human isoforms. It is encoded by an alternative transcript that begins within intron 64 of the VLGR1 gene. The longest gene product, VLGR1b, is 6307 amino acids (6298 amino acids in mice) due to a much larger ectodomain containing 35 calcium exchanger beta repeats and a pentraxin homology domain. VLGR1b is apparently the largest known cell surface protein. The VLGR1 gene comprises 90 exons and is >600 kb long. In situ hybridization studies with mouse embryo sections show that high level expression of VLGR1 is restricted to the developing central nervous system and eye. Strong expression in the ventricular zone, home of neural progenitor cells during embryonal neurogenesis, suggests a fundamental role for VLGR1 in the development of the central nervous system.