The selective sphingosine 1-phosphate receptor modulator BAF312 redirects lymphocyte distribution and has species-specific effects on heart rate.

BACKGROUND AND PURPOSE: BAF312 is a next-generation sphingosine 1-phosphate (S1P) receptor modulator, selective for S1P(1) and S1P(5 ) receptors. S1P(1) receptors are essential for lymphocyte egress from lymph nodes and a drug target in immune-mediated diseases. Here, we have characterized the immunomodulatory potential of BAF312 and the S1P receptor-mediated effects on heart rate using preclinical and human data. EXPERIMENTAL APPROACH: BAF312 was tested in a rat experimental autoimmune encephalomyelitis (EAE) model. Electrophysiological recordings of G-protein-coupled inwardly rectifying potassium (GIRK) channels were carried out in human atrial myocytes. A Phase I multiple-dose trial studied the pharmacokinetics, pharmacodynamics and safety of BAF312 in 48 healthy subjects. KEY RESULTS: BAF312 effectively suppressed EAE in rats by internalizing S1P(1) receptors, rendering them insensitive to the egress signal from lymph nodes. In healthy volunteers, BAF312 caused preferential decreases in CD4(+) T cells, T(naïve) , T(central memory) and B cells within 4-6 h. Cell counts returned to normal ranges within a week after stopping treatment, in line with the elimination half-life of BAF312. Despite sparing S1P(3) receptors (associated with bradycardia in mice), BAF312 induced rapid, transient (day 1 only) bradycardia in humans. BAF312-mediated activation of GIRK channels in human atrial myocytes can fully explain the bradycardia. CONCLUSION AND IMPLICATIONS: This study illustrates species-specific differences in S1P receptor specificity for first-dose cardiac effects. Based on its profound but rapidly reversible inhibition of lymphocyte trafficking, BAF312 may have potential as a treatment for immune-mediated diseases.

The circadian cycle of mPER clock gene products in the suprachiasmatic nucleus of the siberian hamster encodes both daily and seasonal time.

The circadian clock in the hypothalamic suprachiasmatic nuclei (SCN) regulates the pattern of melatonin secretion from the pineal gland such that the duration of release reflects the length of the night. This seasonally specific endocrine cue mediates annual timing in photoperiodic mammals. The aim of this study was to investigate how changes in photoperiod influence the cyclic expression of recently identified clock gene products (mPER and mTIM) in the SCN of a highly seasonal mammal, the Siberian hamster (Phodopus sungorus). Immunocytochemical studies indicate that the abundance of both mPER1 and mPER2 (but not mTIM) in the SCN exhibits very pronounced, synchronous daily cycles, peaking approximately 12 h after lights-on. These rhythms are circadian in nature as they continue approximately under free-running conditions. Their circadian waveform is modulated by photoperiod such that the phase of peak mPER expression is prolonged under long photoperiods. mPER1 protein is also expressed in the pars tuberalis of Siberian hamsters. In hamsters adapted to long days, the expression of mPER1 is elevated at the start of the light phase. In contrast, there is no clear elevation in mPER1 levels in the pars tuberalis of hamsters held on short photoperiods. These results indicate that core elements of the circadian clockwork are sensitive to seasonal time, and that encoding and decoding of seasonal information may be mediated by the actions of these transcriptional modulators.

Combination of non-hypotensive doses of valsartan and enalapril improves survival of spontaneously hypertensive rats with endothelial dysfunction.

There is increasing evidence to suggest endothelial dysfunction as a critical factor in vascular diseases. Genetically predisposed spontaneously hypertensive rats (SHR) treated with inhibitors of nitric oxide (NO) synthase, develop a severe hypertensive nephrosclerosis without the necessity for surgical reduction in renal mass, nephrectomy, renal infarction or nephrotoxic drugs. In these animals, endothelial dysfunction is considered a valid model for assessment of the efficacy of cardiovascular therapy. SHR were treated with either the angiotensin-converting enzyme inhibitor enalapril or the angiotensin II (Ang II) AT(1)-receptor antagonist (AIIA) valsartan at sub-hypotensive doses and the effects on survival rates, cardiac and renal changes were monitored. Rats treated with valsartan, alone or in combination with enalapril, showed markedly higher survival rates (67-85%, respectively) than untreated animals (37%) or those treated with enalapril alone (55%). Valsartan at a dose which attenuated blood pressure increase led to even greater survival rates (95%). Despite these improved survival rates, at non-hypotensive doses the drugs had no effect on histological appearance, nor was kidney function improved. Plasma creatinine levels were reduced by valsartan, alone or in combination with enalapril, but proteinuria persisted with all treatments over the 12 weeks of the study. Aldosterone levels were significantly reduced by all treatments. The results suggest a beneficial role for endothelium in hypertension. Reduced renal perfusion pressure probably underlies the beneficial renal effects of high-dose valsartan.

Mutation in the leptin receptor (Leprfa) causes fat-storage-independent hyperleptinaemia in neonatal rats.

The plasma leptin concentration adjusted for fat mass is affected by mutant gene dosage in older animals segregating for Leprfa. Because the plasma of neonatal rats contains leptin, although their adipocytes contain virtually no triglyceride, we determined whether mutation dose-dependent differences in plasma leptin concentration exist before the postnatal onset of triglyceride storage. Plasma samples were obtained 10 min after birth of each rat pup and leptin concentration determined by radioimmunoassay. Plasma leptin in homozygous wild-type (+/+) pups was 1.6 +/- 0.2 ng/ml (n = 20) and 2.4 +/- 0.2 ng/ml in +/fa (n = 32) littermates (least-square means +/- SE, P < 0.05, two-way ANOVA with litter and genotype as factors). The corresponding values for +/fa (n = 21) and fa/fa (n = 15) littermates were 2.4 +/- 0.2 and, 4.0 +/- 0.3 ng/ml respectively (P < 0.001). Leprfa gene dose-dependent elevations in plasma leptin are, therefore, present at birth and constitute the only Leprfa-related phenotypic trait presently known to precede the onset of increased fat storage.

Leptin responsiveness and gene dosage for leptin receptor mutation (fa) in newborn rats.

To determine the degree to which the leptin receptor mutation (fa) influences the responsiveness to leptin during the first postnatal week, we injected recombinant leptin (600 pmol. g-1. day-1 sc from day 1 to day 7) into wild-type (+/+), heterozygous (+/fa), and fatty (fa/fa) rat pups. Growth and final body fat content of these leptin-treated pups were compared with those of saline-treated littermates of the same genotype. The body mass of the leptin-treated +/+ pups, but not that of the +/fa and fa/fa pups, increased more slowly than that of their respective controls, and fat content at day 7 was reduced by 37% in +/+ pups, by 22% in +/fa pups, but not at all in fa/fa pups. Plasma leptin remained excessively high throughout the day under this treatment, but a 30-fold lower leptin dose, causing only moderate changes of plasma leptin, still reduced the body fat of +/+ pups significantly. We conclude that leptin participates in the control of even the earliest stages of fat deposition and that the response to supraphysiological doses of leptin is markedly reduced in 1-wk-old pups with one fa allele and absent in pups with two fa alleles.

Leptin selectively increases energy expenditure of food-restricted lean mice.

OBJECTIVE: To find out whether leptin can attenuate hypometabolic torpor-like states of metabolic rate (MR) in adult lean animals, as it attenuates the morning suppression of thermoregulatory thermogenesis in suckling-age rat pups. DESIGN: Leptin effects on MR and food intake were studied in mice aged 4-7 months, in which a high incidence of exaggerated circadian reductions of MR had been induced by chronic food-restriction and, for comparison, in free-feeding mice. PROTOCOL: Continuous recordings of MR, for a group of seven mice maintained at an ambient temperature of 24 degrees C, while they were repeatedly-with pauses of at least six days-treated for three consecutive days with either recombinant murine leptin (20, 200 or 600 pmol x g(-1) x d[-1]) or saline. RESULTS: Leptin treatment caused dose-dependent 5-15% increases in energy expenditure by moderating the decreases in MR during the circadian minima, without affecting either the MR during the circadian maxima or food intake. Similar treatment of free-feeding mice caused dose-dependent decreases of food intake without changing MR. CONCLUSION: Leptin controls thermoregulatory energy expenditure when food supplies are scarce and changes food intake, rather than energy expenditure, when food is abundant.

The leptin receptor mediates apparent autocrine regulation of leptin gene expression.

The possibility that the leptin receptor (LEPR) mediates autocrine regulation of leptin expression in adipose tissue was examined in 10-day-old Zucker rat pups with different copy numbers of the leptin receptor mutation (Lepr(fa)). Plasma leptin concentrations and adipose tissue mRNA levels for leptin were related to copy number of the mutation (fa/fa > fa/+ > +/+). These relationships were independent of plasma insulin concentration. Reduced copy number for the functional leptin receptor apparently results in a diminished negative feedback signal to the leptin gene in adipose tissue. Thus, leptin appears to close a short regulatory loop controlling its own synthesis in adipose tissue.

Leptin does not reduce body fat content but augments cold defense abilities in thermoneutrally reared rat pups.

A previous study showed that recombinant leptin markedly affects the body fat content and thermoregulatory energy expenditure of lean (+/+ and +/fa) suckling-age rats, and we wanted to find out whether leptin in doses that halved body fat of cold-reared lean pups had any effect in thermoneutrally reared lean pups. When +/+ pups were artificially reared from 4 to 16 days of age at thermoneutrality and treated as before with leptin from day 7, their total metabolic rate throughout the treatment period was only 4% higher than that of the control littermates and their final body fat content only 4% lower (both P>0.05). We conclude from comparisons of the results in +/+ pups at cold and thermoneutral conditions that leptin does not stimulate, but rather disinhibits, sympathetically mediated thermoregulatory thermogenesis.

Manipulation of potential perinatal zeitgebers for the juvenile circadian temperature rhythm in rats.

We investigated the importance of pre- and postnatal maternal rhythmicity for the development and synchronization of the juvenile circadian core temperature (Tc) rhythm in rats by evaluating the Tc of artificially reared pups in six litters derived from mothers maintained in continuous bright light (LL) and impregnated after drinking behavior stopped showing circadian periodicity. Pups removed from their aperiodic mothers on postnatal day 9 and artificially reared for 3 days showed a free-running Tc rhythm whose amplitude (3.1 +/- 0.1 degrees C, n = 47) was only slightly smaller than that of control pups born to mothers maintained in a 12:12-h light-dark cycle. In four litters the acrophases were not much less synchronized than in the four control litters (mean vector lengths 0.79 +/- 0.05 vs. 0.94 +/- 0.04). In two litters synchronization among littermates was not significant. Additional experiments with cross-fostered pups showed that synchronization is not caused by the time of birth. We conclude that synchronization among littermates can develop even when maternal rhythmicity has been suppressed even before conception, possibly because the Tc decrease of several littermates happening to have similar phases at birth acts as zeitgeber.

Pronounced juvenile circadian core temperature rhythms exist in several strains of rats but not in rabbits.

Torpor-like circadian variations of core temperature are well documented for suckling-age Zucker rat pups. To determine (1) whether this juvenile circadian rhythm is as strongly expressed in other rat strains, and (2) whether a similar rhythm is expressed in rabbit pups, we recorded core temperature and metabolic rate of artificially reared pups. Wistar, Brown Norway, and Long Evans pups were studied for 30 h under moderate cold loads (ambient temperature = 28 degrees C) when 9-11 days old, i.e. at the age and ambient temperature for which the rhythm has been most thoroughly characterized in Zucker rats. Chinchilla bastard rabbit pups were studied under similar conditions when they were 3-8 days old, the youngest age at which the rhythm can be easily detected in rats. Rat pups of each strain showed clear circadian rhythms with sharp decreases of core temperature and metabolic rate in subjective morning. Core temperature amplitudes were in the order Wistar < Brown Norway < Zucker < Long Evans strain. In contrast, the rabbit pups maintained stable high levels of core temperature and metabolic rate throughout the day. A torpor-like decrease of core temperature in the morning is thus not a peculiarity of the Zucker rat strain but also occurs in other pigmented rat strains, whereas rabbit pups at a similar developmental stage do not show a circadian core temperature rhythm.

Is the circadian core temperature rhythm of juvenile rats due to a periodic blockade of thermoregulatory thermogenesis?

Previous studies have demonstrated an endogenous circadian rhythm of core temperature (Tc) in suckling-age rat pups. Our aim was to establish whether the low and irregular Tc at the beginning of the light phase is caused by a temporary blockade of thermoregulatory thermogenesis. We therefore isolated and artificially fed 10-day-old pups for 30 h at five ambient temperatures (Ta), ranging from thermoneutrality to severe cold loading, while continuously recording Tc and metabolic rate (MR). During the maximum phase of the Tc rhythm MR increased linearly with decreasing Ta--to as much as 260% of the daily mean MR at thermoneutrality (TNMR)--so that Tc decreased less than 1 degree C with increasing cold load. During the minimum phase, the MR at all but the lowest Ta fell to, or even below, the TNMR and the amplitude of the Tc rhythm increased from 2 to 5 degrees C with increasing cold load. Under the most severe cold load, however, a further decrease of the minimum Tc was prevented by an increase of MR to 135% of the TNMR. Supplementing the continuously fed synthetic milk with noradrenaline (1.2 mg kg-1 h-1) during the minimum phase of the Tc rhythm increased MR upto 290% of the TNMR. The loose regulation of Tc during the minimum phase of the juvenile circadian Tc rhythm is thus not caused by a peripheral impairment of thermogenic capacity at the beginning of the light phase.