Immunohistochemical localization of D-alanine to beta-cells in rat pancreas

A mouse monoclonal antibody against D-alanine (D-Ala) has been raised and the immunohistochemical localization of this D-amino acids in the rat pancreas is visualized. The obtained anti-D-Ala monoclonal antibody has no significant cross-reactivity to all proteinogenic L-amino acids and their D-enantiomers. Using this antibody, immunohistochemical staining was performed on the pancreas, and specific staining for d-Ala has been observed only in the Langerhans islets. To identify the types of D-Ala-immunopositive cells, double staining was carried out with antibodies against D-Ala and pancreatic hormones. Similar immunostaining patterns have been observed for D-Ala and insulin, while D-Ala is hardly co-localized with other hormones (glucagon, somatostatin, and pancreatic polypeptide). These results indicate for the first time that D-Ala is localized to insulin producing beta-cells in mammalian pancreas, suggesting that this D-amino acid would be involved in the regulation of the blood glucose level.     

Period responses to Zeitgeber signals stabilize circadian clocks during entrainment

Circadian clocks with characteristic period (τ) can be entrained to light/dark (LD) cycles by means of (i) phase shifts which are due to D/L “dawn” and/or L/D “dusk” transitions, (ii) period changes associated with long-term light exposure, or (iii) by combinations of the above possibilities. Based on stability analysis of a model circadian clock it was predicted that nocturnal burrowing mammals would benefit less from period responses than their diurnal counterparts. The model further predicted that maximal stability of circadian clock is reached when the clock slightly changes both its phase and period in response to light stimuli. Analyses of empirical phase response curve (PRC) and period response curve (τRC) of some diurnal and nocturnal mammals revealed that PRCs of both diurnal and nocturnal mammals have similar waveform while τRCs of nocturnal mammals are of smaller amplitude than those of diurnal mammals. The shape of the τRC also changes with age and with increasing strength of light stimuli. During erratic fluctuations in light intensity under different weather conditions, the stability of phase of entrainment of circadian clocks appears to be achieved by an interplay between phase and period responses and the strength of light stimuli.     

Daily rhythms of Fos expression in hypothalamic targets of the suprachiasmatic nucleus in diurnal and nocturnal rodents.

Little is known about the differences in the neural substrates of circadian rhythms that are responsible for the maintenance of differences between diurnal and nocturnal patterns of activity in mammals. In both groups of animals, the suprachiasmatic nucleus (SCN) functions as the principal circadian pacemaker, and surprisingly, several correlates of neuronal activity in the SCN show similar daily patterns in diurnal and nocturnal species. In this study, immunocytochemistry was used to monitor daily fluctuations in the expression of the nuclear phosphoprotein Fos in the SCN and in hypothalamic targets of the SCN axonal outputs in the nocturnal laboratory rat and in the diurnal murid rodent, Arvicanthis niloticus. The daily patterns of Fos expression in the SCN were very similar across the two species. However, clear species differences were seen in regions of the hypothalamus that receive inputs from the SCN including the subparaventricular zone. These results indicate that differences in the circadian system found downstream from the SCN contribute to the emergence of a diurnal or nocturnal profile in mammals.     

The effects of melatonin and L-DOPA on the diurnal rhythms of free amino acids content in the rat retina

The effects of melatonin and dopamine precursor L-3,4-dihydroxyphenylalanine (L-DOPA) intraperitoneal administration on the rhythms of free amino acids content in the retina of rats were studied. The authors found that the levels of those amino acids, which are protein constituents but not neurotransmitters in the rat retina, change diurnally with maximum at 3-6 h after light onset. Diurnal changes of Ala, Arg, Asn, Ile, Met, Ser, Trp, and Val content persisted in the retina of rats maintained at constant darkness. This fact confirms the true circadian nature of these rhythms. Constant lighting abolished diurnal changes of the content of all amino acids with the exception of Trp. Daytime but not nighttime administration of melatonin decreased the levels of Ala, Asn, Gln, Ile, Met, and Ser down to nocturnal values. Diurnal changes of amino acids content vanished in melatonin-injected rats. The effect of melatonin administration disappeared when the protein synthesis was inhibited by cycloheximide. The effect of intraperitoneal administration of L-DOPA on the levels of free amino acids was opposite the effect of melatonin administration. L-DOPA increased nocturnal levels of Gly, Thr, Trp, and Val but had no effect on the daytime amino acids content. As in the case of melatonin administration, significant diurnal changes of amino acid levels disappeared in L-DOPA-injected rats. The authors hypothesize that melatonin and dopamine can serve as zeitgebers-antagonists of amino acids content rhythms in the rat retina.     

Novel phase-shifting effects of GABAA receptor activation in the suprachiasmatic nucleus of a diurnal rodent

The vast majority of neurons in the suprachiasmatic nucleus (SCN), the primary circadian pacemaker in mammals, contain the inhibitory neurotransmitter GABA. Most studies investigating the role of GABA in the SCN have been performed using nocturnal rodents. Activation of GABA(A) receptors by microinjection of muscimol into the SCN phase advances the circadian activity rhythm of nocturnal rodents, but only during the subjective day. Nonphotic stimuli that reset the circadian pacemaker of nocturnal rodents also produce phase advances during the subjective day. The role of GABA in the SCN of diurnal animals and how it may differ from nocturnal animals is not known. In the studies described here, the GABA(A) agonist muscimol was microinjected directly into the SCN region of diurnal unstriped Nile grass rats (Arvicanthis niloticus) at various times in their circadian cycle. The results demonstrate that GABA(A) receptor activation produces large phase delays during the subjective day in grass rats. Treatment with TTX did not affect the ability of muscimol to induce phase delays, suggesting that muscimol acts directly on pacemaker cells within the SCN. These data suggest that the circadian pacemakers of nocturnal and diurnal animals respond to the most abundant neurochemical signal found in SCN neurons in opposite ways. These findings are the first to demonstrate a fundamental difference in the functioning of circadian pacemaker cells in diurnal and nocturnal animals.     

Circadian rhythms of food intake, plasma glucose and insulin levels in fed and fasted rabbits.

The rhythm of circadian variations of plasma insulin level was similar in 48 h fasted and in fed rabbits; however, the amplitude of variations was less important in fasted animals. Plasma glucose level did not change during circadian cycle. In conclusion, we showed in rabbit a circadian rhythm of insulin with two maxima: one diurnal and the other nocturnal.     

Circadian regulation of gonadotropin-releasing hormone neurons and the preovulatory surge in luteinizing hormone in the diurnal rodent, Arvicanthis niloticus, and in a nocturnal rodent, Rattus norvegicus

Daily rhythms in the timing of the preovulatory surge and the display of reproductive behavior are reversed in diurnal and nocturnal rodents, but little is known about the neural mechanisms underlying these differences. We examined this issue by comparing a diurnal murid rodent, Arvicanthis niloticus (the grass rat), to a nocturnal one, Rattus norvegicus (the lab rat). In the first study, we established that sequential estradiol and progesterone treatment induces a proestrous-like rise in LH secretion and in the percentage of GnRH neurons that express Fos in grass rats, as is the case in lab rats. Next, we tested the hypothesis that differences in the timing of estrus-related events in diurnal and nocturnal species are caused by differences in rhythms in responsiveness to steroid hormones. We found rhythms in GnRH neuron activity, as indicated by Fos, that were 12 hours out of phase in grass rats and lab rats. These patterns persisted in both species when animals were housed in constant darkness for 5 days, suggesting that they are driven by an endogenous circadian mechanism. These results indicate that steroid-primed grass rats and lab rats are similar with respect to the temporal relationship among estrus-related events, but that the timing of these events relative to the light-dark cycle is dramatically different and that this difference is caused by endogenous circadian mechanisms.     

Circadian variation in renal function of the obese rat.

The influence of food and water intake on renal function was assessed by comparisons between the hyperphagic Zucker obese rat and its lean littermate, which demonstrates nocturnal dominance in activity. Serum creatinine and cortisol levels, creatine kinase activities, creatinine and urine clearances, and sodium and potassium excretion rates were measured over a 24-hour period in both lean and obese rats (n = 24 each). Six rats in each group were studied every 8 h to permit characterization over a 12-hour light/dark cycle at 2-hour intervals. Urine and creatinine clearances were increased in lean rats during the dark phase coincident with onset of eating. Similarly, renal sodium and potassium excretion rates were markedly increased during the dark cycle, despite relatively constant serum potassium and sodium levels over the 24-hour period. In contrast, no circadian patterns in urine and creatinine clearances were found in the obese rat, which exhibits continuous feeding habits throughout the 24-hour period. Moreover, renal electrolyte excretion in the obese rat was modestly increased during the dark cycle, unlike the significant differences over time observed in lean rats. Serum creatinine levels were increased during the dark cycle in both rat groups. Creatine kinase activity, a measure of ambulatory activity, was constant in lean rats during the study period. Although creatine kinase activity was increased in obese rats during the dark cycle, no correlations with renal functional parameters were found. These results indicate that differences in food and water intake are significant determinants in diurnal cyclic changes in renal function.     

Defective diurnal changes of food intake, plasma glucose and insulin in rats with a transplantable islet cell tumour

Subcutaneous implantation of small fragments of a radiation-induced transplantable rat insulinoma into the subscapular region of 16- to 17-week-old male NEDH rats resulted, over a 22-day period, in the progressive development of marked hyperinsulinaemia and severe hypoglycaemia, despite a compensatory increase in food intake. Diurnal changes were examined at 3-hourly intervals for 24 h in control rats and tumour-bearing rats at 20-21 days after transplantation. The control animals exhibited distinct diurnal changes of food intake, glucose and insulin concentrations. Food intake was greatest between 17.00 and 23.00 h; plasma insulin was greatest between 20.00 and 23.00 h, and plasma glucose was raised at 20.00, 02.00 and 05.00 h, compared with the other times. In contrast, insulinoma-bearing rats displayed no diurnal changes other than a small decrease in food intake between 05.00 and 11.00 h. Plasma glucose and insulin concentrations were significantly different from control rats at all times, and food intake was significantly increased between 23.00 and 17.00 h. These observations demonstrate that the transplantable insulinoma not only causes hyperinsulinaemia and hypoglycaemia but results in hyperphagia and defective diurnal changes of food intake, plasma glucose and insulin concentrations. Interruption of nutrient intake by withdrawal of food for 6 h exacerbated the hypoglycaemia of insulinoma-bearing rats leading to coma.     

Mammalian diurnality: some facts and gaps

A major factor contributing to the evolution of mammals was their ability to be active during the night, a niche previously underused by terrestrial vertebrates. Diurnality subsequently reemerged multiple times in a variety of independent lineages. This paper reviews some recent data on circadian mechanisms in diurnal mammals and considers general themes that appear to be emerging from this work. Careful examination of behavioral studies suggests that although subtle differences may exist, the fundamental functions of the circadian system are the same, as seems to be the case with respect to the molecular mechanisms of the clock. This suggests that responses to signals originating in the clock must be different, either within the SCN or at its targets or downstream from them. Some features of the SCN vary from species to species, but none of these has been clearly associated with diurnality. The region immediately dorsal to the SCN, which receives substantial input from it, exhibits dramatically different rhythms in nocturnal lab rats and diurnal grass rats. This raises the possibility that it functions as a relay that transforms the signal emitted by the SCN and transmits different patterns to downstream targets in nocturnal and diurnal animals. Other direct targets of the SCN include neurons containing orexin and those containing gonadotropin-releasing hormone, and both of these populations of cells exhibit patterns of rhythmicity that are inverted in at least one diurnal compared to one nocturnal species. The patterns that emerge from the data on diurnality are discussed in terms of the implications they have for the evolution and neural substrates of a day-active way of life.     

Photic and nonphotic inputs to the diurnal circadian clock

Diurnal animals occupy a different temporal niche from nocturnal animals and are consequently exposed to different amounts of light as well as different dangers. Accordingly, some variation exists in the way that diurnal animals synchronize their internal circadian clock to match the external 24-hour daily cycle. First, though the brain mechanisms underlying photic entrainment are very similar among species with different daily activity patterns, there is evidence that diurnal animals are less sensitive to photic stimuli compared to nocturnal animals. Second, stimuli other than light that synchronize rhythms (i.e. nonphotic stimuli) can also entrain and phase shift daily rhythms. Some of the rules that govern nonphotic entrainment in nocturnal animals as well as the brain mechanisms that control nonphotic influences on rhythms do not appear to apply to diurnal animals, however. Some evidence supports the idea that arousal or activity plays an important role in entraining rhythms in diurnal animals, either during the light (active) or dark (inactive) phases, though no consistent pattern is seen. GABAergic stimulation induces phase shifts during the subjective day in both diurnal and nocturnal animals. In diurnal Arvicanthis niloticus (Nile grass rats), SCN GABA_A receptor activation at this time results in phase delays while in nocturnal animals phase advances are induced. It appears that the effect of GABA at this circadian phase results from the inhibition of period gene expression in both diurnal and nocturnal animals. Nonetheless, the resulting phase shifts are in opposite directions. It is not known what stimuli or behaviours ultimately induce changes in GABA activity in the SCN that result in alterations of circadian phase in diurnal grass rats. Taken together, studies such as these suggest that it may be problematic to apply the principles governing nocturnal nonphotic entrainment and its underlying mechanisms to diurnal species including humans.     

In vitro protein synthesis using D-amino acids and its evolutionary significance

The in vitro incorporations of D-Trp or -Ala into peptides were investigated using a crude system. Although none of aminoacylation to the former was observed under the present condition, the latter was utilized for peptide synthesis at the same rate as the opposite enantiomer. The results of analyzing the peptides showed that the peptides, which were synthesized with either D-, L- or DL-Ala as the substrate, were composed of mostly similar molecular size. The study of incorporations of radioisotopes (14C-D- and 3H-L-Ala) into peptides also indicated that D-Ala was by no means inferior to L-Ala for the substrate. Basing upon the present experiment, the evolutionary significance for utilizing D-amino acids is discussed.     

The effects of nocturnal life on endocrine circadian patterns in healthy adults

We observed the 24-hour patterns of endocrine in medical students who lived either a diurnal life or nocturnal life. Nocturnal life was designed by skipping their breakfast but consuming much (>50% of their daily food intake) in the evening and at night with the sleep from 0130 h to 0830 h the next morning. After 3 weeks in the experimental life, the 24-hour plasma concentrations of melatonin, leptin, glucose and insulin were measured every three hours. Both plasma melatonin and leptin showed peaks at 0300 h in the diurnal lifestyle group, and the night peaks decreased in the nocturnal lifestyle group. The changes in the patterns of melatonin and leptin were highly consistent with that of night-eating syndrome (NES). Plasma glucose increased after all meals in both groups. Its concentration maintained a high level in the nocturnal lifestyle group between midnight and early morning while insulin secretion decreased markedly during this period. Furthermore, the strong association between glucose and insulin in the diurnal lifestyle group after meals was damaged in the nocturnal lifestyle group. It was suggested that nocturnal life leads to the impairment of insulin response to glucose. Taking these results together, nocturnal life is likely to be one of the risk factors to health of modern people, including NES, obesity and diabetes.     

Cloning and functional characterization of Arabidopsis thaliana D-amino acid aminotransferase--D-aspartate behavior during germination

The understanding of D-amino acid metabolism in higher plants lags far behind that in mammals, for which the biological functions of these unique amino acids have already been elucidated. In this article, we report on the biochemical behavior of D-amino acids (particularly D-Asp) and relevant metabolic enzymes in Arabidopsis thaliana. During germination and growth of the plant, a transient increase in D-Asp levels was observed, suggesting that D-Asp is synthesized in the plant. Administration of D-Asp suppressed growth, although the inhibitory mechanism responsible for this remains to be clarified. Exogenous D-Asp was efficiently incorporated and metabolized, and was converted to other D-amino acids (D-Glu and D-Ala). We then studied the related metabolic enzymes, and consequently cloned and characterized A. thaliana D-amino acid aminotransferase, which is presumably involved in the metabolism of D-Asp in the plant by catalyzing transamination between D-amino acids. This is the first report of cDNA cloning and functional characterization of a D-amino acid aminotransferase in eukaryotes. The results presented here provide important information for understanding the significance of D-amino acids in the metabolism of higher plants.     

Circadian variations in plasma growth hormone and prolactin in the infant rat: Comparison with the adult pattern

Radioimmunoassayable (RIA) plasma growth hormone (GH) and prolactin (PRL) levels were determined at 3 hr intervals during a controlled 24-hr light-dark cycle in 10-day-old male and female rats; parallel measurements were made of brain monoamines (MA's), dopamine (DA), norepinephrine (NE) and serotonin (5-HT) concentration. Plasma GH and PRL and brain MA levels found in infant rats were compared to the same determinations made during the 24-hr cycle in 50-day-old male rats. GH levels were rather uniform and did not show circadian periodicity in the plasma of infant rats, while PRL levels showed a diurnal surge in the late afternoon hr (1800). In adult rats, GH levels exhibited wide fluctuations during the 24-hr cycle and no circadian periodicity, while PRL levels showed one diurnal (1500–1800) and one nocturnal (2400) surge. A pulsatile GH secretion was found in adult rats sampled at 15 min intervals over a period of 2 hr, which seemed to be lacking in infant rats. In the brain of infant rats, DA and NE levels exhibited circadian patterns which resembled the ones present in the brain of adult rats, whereas no circadian variations were present in 5-HT levels.     

RHYTHMIC cFOS EXPRESSION IN THE VENTRAL SUBPARAVENTRICULAR ZONE INFLUENCES GENERAL ACTIVITY RHYTHMS IN THE NILE GRASS RAT,ARVICANTHIS NILOTICUS

Circadian rhythms in behavior and physiology are very different in diurnal and nocturnal rodents. A pacemaker located in the suprachiasmatic nucleus (SCN) of the hypothalamus is responsible for generating and maintaining circadian rhythms in mammals, and cellular and molecular rhythms within the SCN of diurnal and nocturnal rodents are very similar. The neural substrates determining whether an animal has a diurnal or nocturnal phase preference are thus likely to reside downstream of the SCN. The ventral subparaventricular zone (vSPVZ), a major target of the SCN that is important for the expression of circadian rhythmicity in nocturnal lab rats (Rattus norvegicus), exhibits different rhythms in cFos expression in diurnal Nile grass rats compared to lab rats. We examined the effects of chemotoxic lesions of the cFos-expressing cells of the vSPVZ on activity rhythms of grass rats to evaluate the hypothesis that these cells support diurnality in this species. Male grass rats housed in a 12:12 light:dark (LD) cycle were given bilateral injections of the neurotoxin n-methyl-D-L-aspartic acid (NMA) or vehicle aimed at the vSPVZ; cells in the SCN are resistant to NMA, which kills neurons in other brain regions, but leaves fibers of passage intact. vSPVZ-damaged grass rats exhibited highly unstable patterns of activity in constant darkness (DD) and in the LD cycle that followed. However, crepuscular bouts of activity could be seen in all animals with vSPVZ lesions. Damage to the vSPVZ reduced cFos expression in this area but not in the SCN. Using correlational analyses, we found that the number of cFos-ir cells in the vSPVZ was unrelated to several parameters of the activity rhythms during the initial post-surgical period, when animals were in LD. However, the number of cells expressing cFos in the vSPVZ was positively correlated with general activity during the subjective day relative to the subjective night when the animals were switched to DD, and this pattern persisted when a LD cycle was reinstated. Also, the number of cFos-ir cells in the vSPVZ was negatively correlated with the strength of rhythmicity in DD and the number of days required to re-entrain to a LD cycle following several weeks in DD. These data suggest that the vSPVZ emits signals important for the expression of stable diurnal activity patterns in grass rats, and that species differences in these signals may contribute to differences in behavioral and physiological rhythms of diurnal and nocturnal mammals. (Author correspondence: mschw009@umaryland.edu)     

Cardiovascular control by the suprachiasmatic nucleus: neural and neuroendocrine mechanisms in human and rat

The risk for cardiovascular incidents is highest in the early morning, which seems partially due to endogenous factors. Endogenous circadian rhythms in mammalian physiology and behavior are regulated by the suprachiasmatic nucleus (SCN). Recently, anatomical evidence has been provided that SCN functioning is disturbed in patients with essential hypertension. Here we review neural and neuroendocrine mechanisms by which the SCN regulates the cardiovascular system. First, we discuss evidence for an endogenous circadian rhythm in cardiac activity, both in humans and rats, which is abolished after SCN lesioning in rats. The immediate impact of retinal light exposure at night on SCN-output to the cardiovascular system, which signals 'day' in both diurnal (human) and nocturnal (rat) mammals with opposite effects on physiology, is discussed. Furthermore, we discuss the impact of melatonin treatment on the SCN and its potential medical relevance in patients with essential hypertension. Finally, we argue that regional differentiation of the SCN and autonomous nervous system is required to explain the multitude of circadian rhythms. Insights into the mechanisms by which the SCN affects the cardiovascular system may provide new strategies for the treatment of disease conditions known to coincide with circadian rhythm disturbances, as is presented for essential hypertension.     

Diurnal variations in food intake and in lipogenesis in mammary gland and liver of lactating rats.

Despite the hyperphagia, the food intake of the lactating rat showed marked diurnal changes which paralleled those of virgin rats. The major difference was that lactating rats consumed a higher proportion (35%) of their diet during the light period than did virgin rats (14%). The peak rate of lipogenesis in the lactating mammary gland occurred around midnight, and this decreased by 67% to reach a nadir around mid-afternoon; this corresponded with the period of lowest food intake. The diurnal variations in hepatic lipogenesis in lactating rats were much less marked. The changes in hepatic glycogen over 24 h suggest that it acts to supply carbon for lipogenesis during the period of decreased food intake. The activation state of acetyl-CoA carboxylase in mammary gland altered during 24 h, but the changes did not always correlate with alterations in the rate of lipogenesis. The changes in plasma insulin concentration tended to parallel the food intake in the lactating rats, but they did not appear to be sufficient to explain the large alterations in lipogenic rate in the mammary gland.