Malfunction of the circadian clock has been linked to the pathogenesis of a variety of diseases. We show that mice lacking the core clock components Cryptochrome-1 (Cry1) and Cryptochrome-2 (Cry2) (Cry-null mice) show salt-sensitive hypertension due to abnormally high synthesis of the mineralocorticoid aldosterone by the adrenal gland. An extensive search for the underlying cause led us to identify type VI 3beta-hydroxyl-steroid dehydrogenase (Hsd3b6) as a new hypertension risk factor in mice. Hsd3b6 is expressed exclusively in aldosterone-producing cells and is under transcriptional control of the circadian clock. In Cry-null mice, Hsd3b6 messenger RNA and protein levels are constitutively high, leading to a marked increase in 3beta-hydroxysteroid dehydrogenase-isomerase (3beta-HSD) enzymatic activity and, as a consequence, enhanced aldosterone production. These data place Hsd3b6 in a pivotal position through which circadian clock malfunction is coupled to the development of hypertension. Translation of these findings to humans will require clinical examination of human HSD3B1 gene, which we found to be functionally similar to mouse Hsd3b6.

Jet-lag symptoms arise from temporal misalignment between the internal circadian clock and external solar time. We found that circadian rhythms of behavior (locomotor activity), clock gene expression, and body temperature immediately reentrained to phase-shifted light-dark cycles in mice lacking vasopressin receptors V1a and V1b (V1a(-/-)V1b(-/-)). Nevertheless, the behavior of V1a(-/-)V1b(-/-) mice was still coupled to the internal clock, which oscillated normally under standard conditions. Experiments with suprachiasmatic nucleus (SCN) slices in culture suggested that interneuronal communication mediated by V1a and V1b confers on the SCN an intrinsic resistance to external perturbation. Pharmacological blockade of V1a and V1b in the SCN of wild-type mice resulted in accelerated recovery from jet lag, which highlights the potential of vasopressin signaling as a therapeutic target for management of circadian rhythm misalignment, such as jet lag and shift work.

The mammalian clock genes, Period and Cryptochrome (Cry), regulate circadian rhythm. We show that circadian rhythmicity and rhythmic expression of Period in the nuclei of inflammatory synovial cells and spleen cells are disturbed in mouse models of experimental arthritis. Expressions of other clock genes, Bmal1 and Dbp, are also disturbed in spleen cells by arthritis induction. Deletion of Cry1 and Cry2 results in an increase in the number of activated CD3+ CD69+ T cells and a higher production of TNF-α from spleen cells. When arthritis is induced, Cry1−/−Cry2−/− mice develop maximal exacerbation of joint swelling, and upregulation of essential mediators of arthritis, including TNF-α, IL-1β and IL-6, and matrix metalloproteinase-3. Wee-1 kinase is solely upregulated in Cry1−/−Cry2−/− mice, in line with upregulation of c-Fos and Wee-1 kinase in human rheumatoid arthritis. The treatment with anti–TNF-α Ab significantly reduced the severity and halted the progression of the arthritis of Cry1−/−Cry2−/− mice and vice versa, ectopic expression of Cry1 in the mouse embryonic fibroblast from Cry1−/−Cry2−/− mice significantly reduced the trans activation of TNF-α gene. Thus, the biological clock and arthritis influence each other, and this interplay can influence human health and disease.

Synchronous oscillations of thousands of cellular clocks in the suprachiasmatic nucleus (SCN), the circadian centre, are coordinated by precisely timed cell–cell communication, the principle of which is largely unknown. Here we show that the amount of RGS16 (regulator of G protein signalling 16), a protein known to inactivate Gαi, increases at a selective circadian time to allow time-dependent activation of intracellular cyclic AMP signalling in the SCN. Gene ablation of Rgs16 leads to the loss of circadian production of cAMP and as a result lengthens circadian period of behavioural rhythm. The temporally precise regulation of the cAMP signal by clock-controlled RGS16 is needed for the dorsomedial SCN to maintain a normal phase-relationship to the ventrolateral SCN. Thus, RGS16-dependent temporal regulation of intracellular G protein signalling coordinates the intercellular synchrony of SCN pacemaker neurons and thereby defines the 24 h rhythm in behaviour.

Lhx9 is a member of the LIM homeobox gene family. It is expressed during mammalian embryogenesis in the brain including the pineal gland. Deletion of Lhx9 results in sterility due to failure of gonadal development. The current study was initiated to investigate Lhx9 biology in the pineal gland. Lhx9 is highly expressed in the developing pineal gland of the rat with transcript abundance peaking early in development; transcript levels decrease postnatally to nearly undetectable levels in the adult, a temporal pattern that is generally similar to that reported for Lhx9 expression in other brain regions. Studies with C57BL/6J Lhx9−/− mutant mice revealed marked alterations in brain and pineal development. Specifically, the superficial pineal gland is hypoplastic, being reduced to a small cluster of pinealocytes surrounded by meningeal and vascular tissue. The deep pineal gland and the pineal stalk are also reduced in size. Although the brains of neonatal Lhx9−/− mutant mice appear normal, severe hydrocephalus develops in about 70 % of the Lhx9−/− mice at 5–8 weeks of age; these observations are the first to document that deletion of Lhx9 results in hydrocephalus and as such indicate that Lhx9 contributes to the maintenance of normal brain structure. Whereas hydrocephalus is absent in neonatal Lhx9−/−mutant mice, the neonatal pineal gland in these animals is hypoplastic. Accordingly, it appears that Lhx9 is essential for early development of the mammalian pineal gland and that this effect is not secondary to hydrocephalus.

Autophagy is a cellular process that nonspecifically degrades cytosolic components and is involved in many cellular responses. We found that amino sugars with a free amino group such as glucosamine, galactosamine and mannosamine induced autophagy via an mTOR-independent pathway. Glucosamine-induced autophagy at concentrations of at least 500 microM to over 40 mM. In the presence of 40 mM glucosamine, autophagy induction was initiated at 6h and reached a plateau at 36 h. Glucosamine-induced autophagy could remove accumulated ubiquitin-conjugated proteins as well as 79-glutamine repeats. Therefore, orally administered glucosamine could contribute to the prevention of neurodegenerative diseases and promotion of antiaging effects.

The 3␤-hydroxysteroid dehydrogenase (3␤-HSD) is an enzyme crucial for steroid synthesis. Two different 3␤-HSD isoforms exist in humans. Classically, HSD3B2 was considered the principal isoform present in the adrenal. However, we recently showed that the alternative isoform, HSD3B1, is expressed specifically within the adrenal zona glomerulosa (ZG), where aldosterone is produced, raising the question of why this isozyme needs to be expressed in this cell type. Here we show that in both human and mouse, expression of the ZG isoform 3␤-HSD is rapidly induced upon angiotensin II (AngII) stimulation. AngII is the key peptide hormone regulating the capacity of aldosterone synthesis. Using the human adrenocortical H295R cells as a model system, we show that the ZG isoform HSD3B1 differs from HSD3B2 in the ability to respond to AngII. Mechanistically, the induction of HSD3B1 involves de novo protein synthesis of the nuclear orphan receptors NGFIB and NURR1. The HSD3B1 promoter contains a functional NGFIB/NURR1-responsive element to which these proteins bind in response to AngII. Knockdown of these proteins and overexpression of a dominant negative NGFIB both reduce the AngII responsiveness of HSD3B1. Thus, the AngII-NGFIB/NURR1 pathway controls HSD3B1. Our work reveals HSD3B1 as a new regulatory target of AngII.T he enzyme 3␤-hydroxysteroid dehydrogenase/⌬ 5 -⌬ 4 -isomerase (3␤-HSD) is essential for the biosynthesis of all active steroid hormones, including those secreted from the adrenal gland (1-4). Whereas two distinct 3␤-HSD isoforms (type I 3␤-HSD, which is encoded by HSD3B1, and type II 3␤-HSD, which is encoded by HSD3B2) exist in humans, it has long been thought that type II 3␤-HSD was the only isoform expressed in the human adrenal glands (1). However, this canonical view was recently revised due to the observation that the alternative isoform, HSD3B1, is expressed within zona glomerulosa (ZG) cells (5, 6), where aldosterone is produced. Interestingly, the mouse also has two isoforms in the adrenal: one (Hsd3b1) is ubiquitous in the cortex, but the other (Hsd3b6) is ZG specific (6-9). Thus, in both species, the adrenals possess a ZG-specific isoform, in addition to the ubiquitous one. However, it remains unknown why these different enzymes are expressed simultaneously in ZG cells. Since these isozymes catalyze the same enzymatic reaction, the question remains open why the newly identified ZG-specific isoform needs to be expressed in ZG cells.Angiotensin II (AngII) is the key peptide hormone in the renin-angiotensin-aldosterone system (RAAS). AngII is a potent secretagogue of the mineralocorticoid aldosterone, which is principally synthesized within the adrenal gland ZG cells through a series of enzymatic reactions that involve multiple enzymes, including 3␤-HSDs. The actions of AngII on ZG cells are often divided into two temporally different phases (10-12): (i) an early (within minutes after stimulation) upregulation of aldosterone synthesis through posttranslational activation of steroidogenic acute regulatory...

Objective: n-3 polyunsaturated fatty acids, especially eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA), have beneficial effects on atherosclerosis. Although specific salutary actions have been reported, the detailed distribution of n-3 polyunsaturated fatty acids in plaque and their relevance in disease progression are unclear. Our aim was to assess the pharmacodynamics of EPA and DHA and their metabolites in atherosclerotic plaques. Approach and Results: Apolipoprotein E-deficient (Apoe −/− ) mice were fed a Western diet supplemented with EPA (1%, w/w) or DHA (1%, w/w) for 3 weeks. Imaging mass spectrometry analyses were performed in the aortic root and arch of the Apoe −/− mice to evaluate the distribution of EPA, DHA, their metabolites and the lipids containing EPA or DHA in the plaques. Liquid chromatography-mass spectrometry and histological analysis were also performed. The intima-media thickness of atherosclerotic plaque decreased in plaques containing free EPA and EPAs attached with several lipids. EPA was distributed more densely in the thin-cap plaques than in the thick-cap plaques, while DHA was more evenly distributed. In the aortic root, the distribution of total EPA level and cholesteryl esters containing EPA followed a concentration gradient from the vascular endothelium to the media. In the aortic arch, free EPA and 12-hydroxy-EPA colocalized with M2 macrophage. Conclusions: Administered EPA tends to be incorporated from the vascular lumen side and preferentially taken into the thin-cap plaque.