LD Animals Research Articles

Evidence of ancestral nocturnality, locomotor clock regression, and cave zone-adjusted sleep duration modes in a cave beetle

The small carrion beetle Ptomaphagus hirtus is an abundant inhabitant of the exceptionally biodiverse Mammoth Cave system. Previous studies revealed negative phototaxis and the expression of biological clock genes in this microphthalmic cave beetle. Here we present results from probing P. hirtus for the entrainment of locomotor rhythms using the TriKinetics activity monitor setup. Although curtailed by low adjustment frequency of animals to the test environment, the data obtained from successfully monitoring two animals in constant darkness (DD) and six animals exposed to 12 hour light-dark cycles (LD) revealed a strong effect of light on locomotor activity in P. hirtus. In LD, activity was prevalent during the artificial night phases while close to absent during the presumptive day phases, suggesting conserved nocturnality. Upon transitioning LD animals to constant darkness, none displayed detectable evidence of free-running activity rhythms, suggesting complete regression of the central circadian clock. Equally notable, overall locomotor activity of the two DD-monitored animals was about three-fold lower compared to LD animals due to longer rest durations in the former. We, therefore, propose the existence of cave zone-specific energy expenditure modes that are mediated through light schedule responsive modification of sleep duration in P. hirtus.

Filgrastim (r-met-hu G-CSF) enhances the efficiency of spermatogenesis in prepubertal Bos indicus bulls.

This study aimed to test the effects of the drug r-met-hu-G-CSF (filgrastim) on spermatogenic efficiency in prepubertal Brahman bulls. Twelve intact, healthy prepubertal bulls were administered 0, 1 (LD=low dose) or 4 (HD=high dose) µg/Kg r-met-hu-G-CSF (daily for 4days), and haematological analysis was performed. Bulls were castrated (D0 or D60). BW (body weight) and SC (scrotal circumference) were recorded. Testis weight and volume were taken at castration with samples for testis histology and stereology: germ cell types, spermatids count and DSP (daily sperm production per gram)/g of testicular parenchyma. Testicular weight, volume, BW, SC and gonadosomatic index (GSI) were NS (LD-HD; p>.05). At D0 (age 11months), the most advanced germ cell types (maGCt) ranged from intermediate spermatogonia to pachytene spermatocytes. After 2months, control animals had round spermatids as maGCt, LD animals 75% round spermatids and 25% elongated spermatids, and HD animals round spermatids. Spermatids/testis were higher in LD (1.23±0.2 millions) than in controls (0.65±0.1 millions, p<.05). Spermatogenic efficiency (DSP/g) was higher in LD (5.4±0.4 million) than in controls (3.2±0.2 million, p<.01). In conclusion, r-met-hu-G-CSF raises spermatogenic efficiency in prepubertal Brahman bulls.

Maternal Chronodisruption Throughout Pregnancy Impairs Glucose Homeostasis and Adipose Tissue Physiology in the Male Rat Offspring

Compelling evidence in rats support the idea that gestational chronodisruption induces major changes in maternal circadian rhythms and fetal development and that these changes impact adult life at many physiological levels. Using a model of chronic photoperiod shifting throughout gestation (CPS), in which pregnant female rats (Sprague–Dawley strain; n = 16 per group) were exposed to lighting schedule manipulation every 3–4 days reversing the photoperiod completely or light/dark photoperiod (12/12; LD), we explored in the adult rat male offspring body weight gain, glucose homeostasis, adipose tissue content, adipose tissue response to norepinephrine (NE), and adipose tissue proteomic in the basal condition with standard diet (SD) and in response to high-fat diet (HFD). In adult CPS male (100–200 days old; n = 8 per group), we found increasing body weight, under SD and adiposity. Also, we found an increased response to intraperitoneal glucose (IGTT). After 12 weeks of HFD, white adipose tissue depots in CPS offspring were increased further, and higher IGTT and lower intraperitoneal insulin tolerance response were found, despite the lack of changes in food intake. In in vitro experiments, we observed that adipose tissue (WAT and BAT) glycerol response to NE from CPS offspring was decreased, and it was completely abolished by HFD. At the proteomic level, in CPS adipose tissue, 275 proteins displayed differential expression, compared with LD animals fed with a standard diet. Interestingly, CPS offspring and LD fed with HFD showed 20 proteins in common (2 upregulated and 18 downregulated). Based on these common proteins, the IPA analysis found that two functional pathways were significantly altered by CPS: network 1 (AKT/ERK) and network 2 (TNF/IL4; data are available via ProteomeXchange with identifier PXD026315). The present data show that gestational chronodisruption induced deleterious effects in adipose tissue recruitment and function, supporting the idea that adipose tissue function was programmed in utero by gestational chronodisruption, inducing deficient metabolic responses that persist into adulthood.

Association of genetic variants and expression levels of porcine FABP4 and FABP5 genes.

The FABP4 and FABP5 genes, coding for fatty acid transport proteins, have long been studied as positional candidate genes for SSC4 QTL affecting fat deposition and composition traits in pigs. Polymorphisms in these genes, FABP4:g.2634_2635insC and FABP5:g.3000T>G, have previously been associated with fatness traits in an Iberian by Landrace cross (IBMAP). The aim of the present work was to evaluate the functional implication of these genetic variants. For this purpose, FABP4 and FABP5 mRNA expression levels in 114 BC1_LD animals (25% Iberian×75% Landrace) were analyzed using real-time quantitative PCR in backfat and muscle. FABP4 gene expression in backfat, but not in muscle, was associated with FABP4:g.2634_2635insC. In contrast, FABP5:g.3000T>G was not associated with gene expression levels. An expression-based genome-wide association study highlighted the FABP4:g.2634_2635insC polymorphism as the polymorphism most associated with FABP4 gene expression in backfat. Furthermore, other genomic regions associated in trans with the mRNA expression of FABP4 in backfat and FABP5 in muscle were also identified. Finally, two putative transcription binding sites for PPARG and NR4A2 may be affected by the FABP4:g.2634_2635insC polymorphism, modifying FABP4 gene expression. Our results reinforce FABP4 as a candidate gene for fatness traits on SSC4.

A global analysis of CNVs in swine using whole genome sequence data and association analysis with fatty acid composition and growth traits.

Copy number variations (CNVs) are important genetic variants complementary to SNPs, and can be considered as biomarkers for some economically important traits in domestic animals. In the present study, a genomic analysis of porcine CNVs based on next-generation sequencing data was carried out to identify CNVs segregating in an Iberian x Landrace backcross population and study their association with fatty acid composition and growth-related traits. A total of 1,279 CNVs, including duplications and deletions, were detected, ranging from 106 to 235 CNVs across samples, with an average of 183 CNVs per sample. Moreover, we detected 540 CNV regions (CNVRs) containing 245 genes. Functional annotation suggested that these genes possess a great variety of molecular functions and may play a role in production traits in commercial breeds. Some of the identified CNVRs contained relevant functional genes (e.g., CLCA4, CYP4X1, GPAT2, MOGAT2, PLA2G2A and PRKG1, among others). The variation in copy number of four of them (CLCA4, GPAT2, MOGAT2 and PRKG1) was validated in 150 BC1_LD (25% Iberian and 75% Landrace) animals by qPCR. Additionally, their contribution regarding backfat and intramuscular fatty acid composition and growth–related traits was analyzed. Statistically significant associations were obtained for CNVR112 (GPAT2) for the C18:2(n-6)/C18:3(n-3) ratio in backfat and carcass length, among others. Notably, GPATs are enzymes that catalyze the first step in the biosynthesis of both triglycerides and glycerophospholipids, suggesting that this CNVR may contribute to genetic variation in fatty acid composition and growth traits. These findings provide useful genomic information to facilitate the further identification of trait-related CNVRs affecting economically important traits in pigs.

P3018 Gene expression analysis in backfat and identification of eQTL regions for fatness and fatty acid composition candidate genes in pigs

Fat content and fatty acid (FA) composition determine important sensory and technological aspects of pork and meat products because of their influence on the melting point and oxidative status of porcine tissues. Artificial selection to increase meat production in pigs has caused a reduction of intramuscular fat and changes in meat FA composition in some breeds. Gene expression levels can be analyzed as a quantitative trait to identify genomic regions regulating its expression. These regions are known as expression quantitative trait loci (eQTLs). The detection of eQTLs has been proposed as a good strategy to deepen the study of the genetic architecture of complex traits. The aim of this work was to study the genetic basis of the backfat expression of 43 lipid-related genes associated with meat quality traits in pigs. We performed an expression genome-wide association (eGWAS) with the backfat gene expression measured by real-time PCR and the PorcineSNP60 BeadChip genotype information in 115 Iberian × Landrace backcross (BC1_LD) animals. The eGWAS identified 224 eSNPs located in 21 chromosomal regions on SSC2-SSC4, SSC6, SSC8-SSC10 and SSC13-SSC15 and associated with the ACSM5, ELOVL6, FABP4, FADS2, and FATP4 genes. Three out of 21 eQTLs corresponding to ACSM5, FABP4, and FADS2 were classified as cis-acting eQTLs, whereas the remaining 18 eQTLs have trans-regulatory effects. In addition, four polymorphisms were identified and genotyped in the BC1_LD animals for the genes with cis-acting eQTLs: two SNPs located in the proximal promoter region of ACSM5 (SSC3) and FADS2 (SSC2), and one Indel and one SNP located in the intron 1 and in the 3’UTR region of FABP4, respectively. It is noteworthy that the strongest association signal for ACSM5 gene expression was shown for the polymorphism located in the promoter region of this gene. For FABP4 gene expression, the SNP in the 3’UTR region showed the lower P-value, and the indel was also one of the most significantly associated, as has been previously described. Thus, these SNPs are strong candidate polymorphisms to explain the mRNA variation of the ACSM5 and FABP4 genes and may have also a role in the determination of the FA composition in the BC1_LD animals. In concordance with these results, FABP4:g.2634_2635insC polymorphism has been associated with the percentage of palmitoleic and eicosatrienoic FA and polyunsaturated FA in muscle. These findings provide resources to decipher the functional regulatory mechanisms implicated in the variation of meat quality traits in pigs.

Progranulin is increased in human and murine lipodystrophy

AimsLipodystrophies (LD) are genetic or acquired disorders sharing the symptom of partial or complete adipose tissue deficiency and a dysregulation of adipokines including leptin and adiponectin. Progranulin, an adipokine with proinflammatory and insulin resistance-inducing characteristics, has not been investigated in LD so far. MethodsCirculating progranulin was determined in LD patients (N=37) and in age-, gender-, and body mass index-matched healthy control subjects (N=37). Additionally, we investigated progranulin expression in an LD mouse model as compared to wild-type mice. Moreover, we elucidated circulating progranulin before and during metreleptin supplementation in 10 patients with LD. ResultsMedian [interquartile range] circulating progranulin was increased in patients with LD (82.9 [25.9] μg/l) as compared to controls (73.6 [22.8] μg/l) (p=0.005). C-reactive protein (CRP) remained an independent and positive predictor of progranulin in multivariate analysis. Progranulin mRNA was significantly upregulated in all adipose tissue depots, i.e. visceral, subcutaneous, and brown adipose tissue, and in muscle of LD animals versus wild-type mice. Progranulin levels did not significantly change during metreleptin supplementation. ConclusionsProgranulin serum concentration is increased in patients with LD, and shows an independent and positive correlation with CRP. Different adipose tissue depots and muscle might be potential origins of elevated progranulin.

Effect of circadian rhythm disturbance on morphine preference and addiction in male rats: Involvement of period genes and dopamine D1 receptor

It is claimed that a correlation exists between disturbance of circadian rhythms by factors such as alteration of normal light–dark cycle and the development of addiction. However, the exact mechanisms involved in this relationship are not much understood. Here we have studied the effect of constant light on morphine voluntary consumption and withdrawal symptoms and also investigated the involvement of Per1, Per2 and dopamine D1 receptor in these processes. Male wistar rats were kept under standard (LD) or constant light (LL) conditions for one month. The plasma concentration of melatonin was evaluated by enzyme-linked immunosorbent assay (ELISA). Real-time PCR was used to determine the mRNA expression of Per1, Per2 and dopamine D1 receptor in the striatum and prefrontal cortex. Morphine preference (50mg/L) was evaluated in a two-bottle-choice paradigm for 10weeks and withdrawal symptoms were recorded after administration of naloxone (3mg/kg). One month exposure to constant light resulted in a significant decrease of melatonin concentration in the LL group. In addition, mRNA levels of Per2 and dopamine D1 receptor were up-regulated in both the striatum and prefrontal cortex of the LL group. However, expression of Per1 gene was only up-regulated in the striatum of LL rats in comparison to LD animals. Furthermore, after one month exposure to constant light, morphine consumption and preference ratio and also severity of naloxone-induced withdrawal syndrome were significantly greater in LL animals. It is concluded that exposure to constant light by up-regulation of Per2 and dopamine D1 receptor in the striatum and prefrontal cortex and up-regulation of Per1 in the striatum and the possible involvement of melatonin makes animals vulnerable to morphine preference and addiction.

Photoperiod induced obesity in the Brandt's vole (Lasiopodomys brandtii): a model of 'healthy obesity'?

ABSTRACTBrandt's voles have an annual cycle of body weight and adiposity. These changes can be induced in the laboratory by manipulation of photoperiod. In the present study, male captive-bred Brandt's voles aged 35 days were acclimated to a short day (SD) photoperiod (8L:16D) for 70 days. A subgroup of individuals (n=16) were implanted with transmitters to monitor physical activity and body temperature. They were then randomly allocated into long day (LD=16L:8D) (n=19, 8 with transmitters) and SD (n=18, 8 with transmitters) groups for an additional 70 days. We monitored aspects of energy balance, glucose and insulin tolerance (GTT and ITT), body composition and organ fat content after exposure to the different photoperiods. LD voles increased in weight for 35 days and then re-established stability at a higher level. At the end of the experiment LD-exposed voles had greater white adipose tissue mass than SD voles (P=0.003). During weight gain they did not differ in their food intake or digestive efficiency; however, daily energy expenditure was significantly reduced in the LD compared with SD animals (ANCOVA, P<0.05) and there was a trend to reduced resting metabolic rate RMR (P=0.075). Physical activity levels were unchanged. Despite different levels of fat storage, the GTT and ITT responses of SD and LD voles were not significantly different, and these traits were not correlated to body fatness. Hence, the photoperiod-induced obesity was independent on disruptions to glucose homeostasis, indicating a potential adaptive decoupling of these states in evolutionary time. Fat content in both the liver and muscle showed no significant difference between LD and SD animals. How voles overcome the common negative aspects of fat storage might make them a useful model for understanding the phenomenon of ‘healthy obesity’.

Abstract T MP16: Impact of Circadian Clock Disruption on Infarct Volume in an Ischemic Stroke Model

Background: At perimenopause, women show increasing incidence of ischemic stroke and poor prognosis for recovery, suggesting that declining ovarian steroid levels alter the risk for neurovascular disease. Peripheral circadian clocks throughout the body may provide integral links between cycling levels of estrogen and neuroprotective responses. Circadian rhythm disruption may be a key factor coupling risk factors (ie, diabetes, obesity) to cardiovascular pathologies. Thus, the present study used an ischemic stroke model to determine whether environmental disruption of circadian rhythms in young adult female rats modulates estrous cyclicity and induces corresponding increases stroke volume and functional deficits similar to that observed in middle-aged females. Methods: Adult (5 mo) female rats were exposed for 8wks to either a fixed or shifted (12hr advance/5d) LD 12:12 cycle and then subjected to middle cerebral artery occlusion (MCAo). Pre and post sensorimotor testing was performed to assess functional deficits. Animals were terminated at 5d post MCAo. Brains processed for histological analysis of infarct volume. Before and after experimental manipulations, estrous cyclicity was assessed via vaginal smears in parallel groups of animals. Results: Circadian activity rhythms stably entrained to the fixed LD cycle but were severely disrupted in shifted LD rats. In contrast to the regular estrous cycles (~7d) in fixed LD animals, cyclicity was abolished and persistent estrous was evident in all shifted LD rats. Total infarct volume in shifted LD rats (0.47) was significantly increased (p&lt;0.05) relative to that observed in fixed LD controls (0.27). Sensorimotor testing revealed a similar trend in which MCAo-induced functional deficits in shifted LD animals were greater than those in fixed LD rats. Conclusion: These results suggest that the loss of estrous cyclicity in young females due to circadian disruption exacerbates stroke outcomes, supporting the hypothesis that in females, middle-age may precipitate circadian disturbances that link reproductive aging to pathological changes in neuroprotective responses to injury. Supported by AHA 14GRNT18370013 to DE

Disturbances in behavior and cortical enkephalin gene expression during the anticipation of ethanol in rats characterized as high drinkers

The process of ethanol anticipation is a particularly important phenomenon that can determine subsequent drug-taking behavior. Recent studies suggest that systems within the medial prefrontal cortex (mPFC), during anticipation, may contribute to the goal-directed seeking of ethanol. The current investigation examined the possibility that the opioid peptide enkephalin (ENK), known to mediate some of the reinforcing properties of ethanol, may function in the mPFC during the anticipation of ethanol access. Using a limited access (3 h/d) paradigm for 10 days with 20% ethanol, Sprague-Dawley rats were first identified either as low drinkers (LD, <1.0 g/kg/3 h) or as high drinkers (HD, >2.0 g/kg/3 h) that exhibited a long-term phenotype of high ethanol consumption and a significant ethanol deprivation effect. During the anticipation period immediately preceding daily ethanol access, the HD rats compared to LD or Control animals with ad libitum ethanol access exhibited increased anticipatory behaviors, including greater exploratory behavior in a novel open field as revealed by significantly more time spent in the rearing position (+53–65%, p < 0.05) and increased number of rears made (+33–44%, p < 0.05) and greater novelty-seeking behavior in a hole-board apparatus revealed by an increase in total (+50–52%, p < 0.05) and novel nose pokes (+45–48%, p < 0.05). In the HD rats, analysis of the mPFC using real-time quantitative PCR showed significantly greater mRNA levels of ENK (p < 0.05) and the mu-opioid receptor (MOR) (p < 0.05), but not delta-opioid receptor (DOR), and this increase in ENK expression was found, using in situ hybridization, to occur specifically in the prelimbic (PrL) subregion of the mPFC. When injected into the PrL during the anticipation period, a MOR agonist but not DOR agonist significantly increased consumption of 20% ethanol (p < 0.05). These findings support the role of ENK, acting through MOR within the PrL to promote the anticipation and excessive consumption of ethanol.

Ovulatory activity and plasma prolactin concentrations in wild and domestic ewes exposed to artificial photoperiods between the winter and summer solstices

Mouflon and domestic Manchega sheep differ in the timing of their reproductive season under natural photoperiod (NP) conditions. This study examines whether they also differ in their reproductive responses to artificial photoperiod cues. For this, mouflons (n=24) and ewes (n=24) were exposed between the winter and summer solstices to artificial long days (LD: 16h light/day), to short days (SD) simulated via the use of melatonin implants, or to NP conditions (controls), and their ovulatory activity monitored. The effects of these treatments on annual changes in prolactin concentration were also recorded. In the LD mouflon ewes, the offset and onset (7 March±5 and 2 October±4, respectively) of cyclic ovulatory activity occurred earlier (P<0.001) than in the NP animals (26 April±6 and 20 October±2, respectively), but no differences were seen (P>0.05) between the SD and NP mouflon ewes in either the onset of anoestrus (12 May±6 and 26 April±6, respectively) or the onset of subsequent ovulatory activity (13 October±8 and 20 October±2, respectively); however the duration of the anoestrus period was significantly reduced in the SD. In LD Manchega ewes, the onset of anoestrus was advanced (2 February±5 vs 15 March±11), but ovulatory activity started at the same time as in NP Manchega ewes (16 July±4 vs 5 July±8). In the SD Manchega ewes, two animals showed continuous cyclic ovulatory activity over the course of the experiment while the remainder entered anoestrus two months later (16 May±6, P<0.001) than their NP counterparts. In these SD ewes, the onset of cyclic ovarian activity was very variable. An annual rhythm of plasma prolactin concentration was seen in both the mouflon and Manchega ewes under all three photoperiod conditions. However, the amplitudes of the changes seen in prolactin concentration were smaller in both the LD and SD animals than in the corresponding NP animals (P<0.001). In conclusion, the results show that these two types of Mediterranean sheep differ in their ovulatory response when subjected to artificial photoperiods. The results also suggest that refractoriness to SDs may be the most important physiological mechanism regulating the onset of anoestrus in highly seasonal breeds, but not in less seasonal breeds.

Effect of season and photoperiod on the time of first postpartum ovulation in Awassi ewes

Seasonal differences in the resumption of postpartum ovarian activity, milk production and periparturient metabolic status were investigated in lactating non-suckling dairy Awassi sheep in two consecutive experiments. In Experiment 1, autumn-lambing (AL, n = 27) and spring-lambing (SL, n = 37) ewes were investigated. Ovarian activity was monitored by means of individual progesterone (P4) profiles from day 5 to day 100 post partum. Most of the AL dams (89%) ovulated till day 35 after parturition and became cyclic thereafter. Incidence of persistent corpus luteum (CLP) and short luteal phases (sCL) was frequent (18% and 29%, respectively) among non-conceiving dams. In contrast, only 24% of the SL ewes ovulated before day 35. P4 levels during the luteal phase were lower in cyclic animals, and the cycle was longer in SL than in AL animals. No CLP or sCL was detected in the spring-lambing group, and 61% of SL ewes remained acyclic till the end of the trial. Lactation length was significantly longer in SL dams than in AL ewes (P = 0.008). According to the plasma metabolites (BHB, NEFA) and metabolic hormones (insulin, IGF-I, thyroxine) examined, negative energy balance did not appear in any of the animals. However, seasonal differences were seen in IGF-I and thyroxine levels, which were higher in the SL dams. In Experiment 2, influence of additional lighting was studied in autumn-lambing ewes. The long-day photoperiod (LD, n = 23) group was exposed to artificial light from sunset till midnight (approx. 16 h light/8 h dark) from some weeks before the expected date of delivery in mid-September until the end of December. The control group (n = 25) experienced only natural daylength. The first postpartum ovulation tended to occur later in the LD animals than in the controls (P = 0.047). The lactation of the LD group tended to be longer (P = 0.061). NEFA, BHB, insulin, IGF-I and thyroxine levels did not differ between the groups. (i) The ovarian function of the Awassi population is seasonal under temperate continental climate conditions. (ii) The first postpartum ovulation of non-suckling, autumn-lambing dams may occur very early, even before the completion of uterine involution. (iii) Additional artificial lighting may delay the time of first postpartum ovulation in AL ewes. (iv) Postpartum negative energy balance is unlikely to occur in dairy Awassi ewes even in high-producing intensive systems.

Poor inhibitory control and neurochemical differences in high compulsive drinker rats selected by schedule-induced polydipsia

Schedule-induced polydipsia (SIP), characterized by the development of excessive drinking under intermittent food reinforcement schedules, has been proposed as a model for obsessive-compulsive disorder, schizophrenia and drug abuse. The purpose of this study is to investigate if individual differences in SIP reflect psychopathological behavioural traits related to lack of inhibitory control and reactivity to novelty, and if these differences have neurochemical correlates. Outbred Wistar rats were selected for being either high (HD) or low (LD) drinkers according to their SIP behaviour. We tested locomotor reactivity to a novel environment and inhibitory control on the five-choice serial reaction time task (5-CSRTT), under baseline vs. extinction conditions and following challenge with D: -amphetamine (saline, 0.5 or 1 mg/kg). Post-mortem analyses of the monoaminergic levels in different brain regions were also analysed. Compared to LD animals, HD rats exhibiting SIP acquisition showed no differences in spontaneous locomotor reactivity to novelty. On the 5-CSRTT, HD rats showed a greater increase in perseverative responses under extinction, a trend towards elevated premature responses on baseline, and a significantly greater elevation of premature responses to D: -amphetamine 0.5 mg/kg. The HD animals also exhibited increased serotonin activity in the amygdala, and correlational analyses between the rate of drinking on SIP and monoamine levels also revealed altered dopaminergic mesolimbic function. These findings show that HD rats selected by SIP exhibit compulsive and impulsive behaviour based on measures of performance on the five-choice serial reaction time task and associated with changes in monoaminergic systems in limbic-striatal circuitry.

Inhibition of matrix metalloproteinases in Siberian hamsters impedes photostimulated recrudescence of ovaries

Exposure of Siberian hamsters to short photoperiod for 14 weeks induces ovarian regression. Subsequent transfer to long photoperiod restores ovarian function, and 2 weeks of photostimulation increases plasma estradiol (E(2)), antral follicles, and corpora lutea (CL). Because tissue remodeling involved with photostimulated ovarian recrudescence is associated with differential expression of matrix metalloproteinases (MMPs), we hypothesized that inhibiting MMP activity using a broad-spectrum in vivo MMP inhibitor, GM6001, would curtail recrudescence. One group of hamsters was placed in long days (LD; 16 h light:8 h darkness) for 16 weeks. Another group was placed in inhibitory short days (SD; 8 h light:16 h darkness) for 14 weeks. A third group was placed in SD for 14 weeks and transferred to LD for 2 weeks to stimulate recrudescence. During weeks 14-16, animals were either not treated or treated daily with i.p. injections of GM6001 (20 mg/kg) or vehicle (DMSO). GM6001 reduced gelatinase activity and decreased immunohistochemical staining for MMP1, MMP2, and MMP3 compared with vehicle. No differences between controls, vehicle, or GM6001 treatment were observed among LD animals, despite a trend toward reduction in CL and E(2) with GM6001. Although SD reduced ovarian function, photostimulation of transferred controls increased uterine mass, plasma E(2), appearance of antral follicles, and CL. With GM6001 treatment, photostimulation failed to increase uterine mass, plasma E(2), antral follicles, or CL. These data show, for the first time, that in vivo GM6001 administration inhibits MMP activity in hamster ovaries during photostimulation, and indicate that this inhibition may impede photostimulated recrudescence of ovaries. This study suggests an intriguing link between MMP activity and return to ovarian function during photostimulated recrudescence.

Constant light induces alterations in melatonin levels, food intake, feed efficiency, visceral adiposity, and circadian rhythms in rats

Melatonin levels, metabolic parameters, circadian rhythm activity patterns, and behavior were observed in rats subjected to a 12-h/12-h light/dark cycle (LD) compared to animals exposed to continuous dark (DD) or continuous light (LL). LD and DD animals were similar in melatonin levels, food intake, relative food intake, feed efficiency, water intake, circadian activity levels, and behavior. LL animals had lower melatonin levels in the subjective dark compared to LD and DD animals. Food intake, relative food intake, and water intake values were lower and feed efficiency was more positive in LL animals compared to LD and DD animals. In addition, LL animals exhibited greater visceral adiposity than the other two groups. The circadian rhythmicity of activity became free-running in LL animals and there was a decrease in overall activity. Notable behavioral changes in LL animals were an increase in irritability and excitability. Results indicate that a decrease in melatonin levels and concomitant changes in metabolism, circadian rhythms, and behavior are consequences of exposure to constant light.

Effects of photoperiod on gluconeogenic activity and total lipid concentration in organs of crabs, Neohelice granulata, challenged by salinity changes

Abstract. This study assessed the effects of long (LD) or short (SD) days on the conversion of [14C]‐glycerol to [14C]‐glucose and total lipid concentration in organs of the crab Neohelice granulata challenged by a change in external salinity. In the 20‰‐acclimated crabs, no difference was found in the concentration of total lipids in the muscle, hepatopancreas, gills, or hemolymph between crabs acclimated to SD or LD. In SD crabs, the total lipid levels in the anterior and posterior gills did not decrease during an osmotic challenge. Only in the posterior gills did the total lipid levels decrease during acclimation to the 34‰ medium in LD animals. The total lipid concentration in the hemolymph decreased after 1 d of osmotic stress in SD, and increased in the hepatopancreas. In LD crabs, the lipid contents decreased gradually in muscle, and in the hepatopancreas on day 3 after transfer to 34‰ medium. In 20‰‐acclimated crabs, the gluconeogenesis activity in both sets of gills was higher in LD than in SD animals. The gluconeogenesis capacity decreased in both sets of gills on the first day of osmotic challenge in SD, and in the posterior gills on the third day in LD crabs. These results suggest that in organs of N. granulata, photoperiod affects the metabolic adjustments to an osmotic challenge.

Proteomic Study of Hepatic Nuclear Extracts in an Adaptive Acetaminophen Tolerance Model

Abstract Introduction Variability in response to acetaminophen (APAP)-induced aseptic inflammation and tolerance to the impending hepatic damage has been described. To understand the mechanism of adaptive tolerance, we investigated the proteomic profiles of crude nuclear lysates in a mouse model. We hypothesized that pretreatment with low doses of APAP prior to a toxic dose results in differential protein expression. Materials and Methods Mice (BALB/C) were separated into three groups: the pretreated (PT) group received incremental doses of APAP while the last dose only (LD) and naïve groups were given saline vehicle. A toxic dose of APAP was administered on the seventh day to the PT and LD animals only and all groups were euthanized 3 h postdose. Total protein from crude hepatic nuclear lysates were applied to protein arrays and analyzed by immunoaffinity mass spectrometry. Results and Discussion Comparative data analyses of protein peaks revealed a protein that was significantly increased at m/z of 60,030 (p60) in the LD animals vs the other two groups. The closest match for the preliminary identification of the p60 protein based on a Swiss-Prot/TagIdent database search using the approximate isoelectric point and molecular weight information was Ccr4–Not complex subunit-2. This protein is a subunit of a multiprotein complex and serves as a transcriptional suppressor involved in controlling mRNA synthesis and degradation. Preliminary identification was also supported by Western blot analysis using anti-CNOT2 antibody. Conclusion Considering the APAP tolerance model, we conclude that toxicogenomic approaches such as nuclear profiling are useful tools in assessing differential expression of transcriptional factors involved in inflammatory response and adaptive tolerance to toxins.

Day‐length encoding through tonic photic effects in the retinorecipient SCN region

The circadian clock in the suprachiasmatic nucleus (SCN) plays a critical role in seasonal processes by sensing ambient photoperiod. To explore how it measures day-length, we assessed the state of SCN oscillators using markers for neuronal activity (c-FOS) and the clock protein (PER1) in Syrian hamsters housed in long (LD, 16 : 8 h light : dark) vs. short days (SD, 8 : 16 h light : dark). During SD, there was no detectable phase dispersion across the rostrocaudal extent of the nucleus. In contrast, during LD, rhythms in the caudal SCN phase led those in the mid- and rostral SCN by 4-8 h and 8-12 h, respectively. Importantly, some neurons in the retinorecipient core SCN were unique in that they were FOS-positive during the dark phase in LD, but not SD. Transfer of LD animals to constant darkness or skeleton photoperiod revealed that dark-phase FOS expression depends on tonic light exposure rather than on intrinsic clock properties. By transferring animals from SD to LD, we next discovered that there are two separate populations of SCN cells, one responding to acute and the other to tonic light exposure. The results suggest that the seasonal encoding of day-length by the SCN entails reorganization of its constituent oscillators by a subgroup of neurons in the SCN core that respond to tonic photic cues.

Gut hormones in relation to body mass and torpor pattern changes during food restriction and re-feeding in the gray mouse lemur

Potential implications of gut hormones in body mass and torpor and behavioral pattern changes induced by an incremental (40 and 80%) calorie restriction (CR) in long-days (LD, summer) and short-days (SD, winter) were investigated in gray mouse lemurs. Only 80% food-deprived LD and SD animals showed a continuous mass loss resulting in a 10 and 15% mass reduction, respectively. Ghrelin levels of all food-deprived groups increased by 2.6-fold on average and remained high after re-feeding while peptide YY (PYY) levels increased by 3.8-fold only in LD animals under 80% CR. In the re-fed SD group, body mass was positively associated with ghrelin and negatively associated with PYY, while no correlations were noted in the re-fed LD animals. Plasma glucagon-like peptide-1 (GLP-1) increased by 2.9-fold only in LD food-restricted mouse lemurs and was negatively associated with the minimal body temperature. No significant correlations were reported in food-deprived SD animals. These results suggest that ghrelin, PYY and GLP-1 may be related to pre-wintering fattening mechanisms and to the modulation of torpor expression, respectively. Such observation clearly warrants further investigations, but it opens an interesting area of research in torpor regulation.