Resting metabolic rate, abdominal fat pad and liver metabolic gene expression in female rats provided a snacking diet from weaning to adulthood.

Our female rat model with continuous, ad libitum access to snacks and chow from weaning to adulthood closely mimics human feeding behavior from childhood onwards. It causes weight gain, enlarged abdominal fat pads, reduced insulin sensitivity and leptin resistance without an increase in total caloric intake. Our current study investigated if this change in energy partitioning is due to a decrease in resting metabolic rate (RMR). In addition, we determined if carbohydrate and lipid metabolism changes in abdominal fat pads and liver. RMR, using indirect calorimetry, was determined in control and snacking rats every two weeks from Days 28-29 to Days 76-77. RMR decreased with age in both groups, but there was no difference between snacking and control rats at any age. At termination, abdominal fat pads (parametrial, retroperitoneal and mesenteric) and liver samples were collected for determination of gene expression for 21 genes involved in carbohydrate and lipid metabolism using RT-qPCR. Analysis of gene expression data showed a striking difference between metabolic profiles of control and snacking rats in abdominal fat pads and liver, with a distinct segregation of genes for both lipid and carbohydrate metabolism that correlated with an increase in body weight and fat pad weights. Genes involved in lipogenesis were upregulated in abdominal fat pads, while genes involved in adipogenesis, and lipid recycling were upregulated in the liver. In conclusion, snacking in addition to chow from weaning in female rats causes a repartitioning of energy that is not due to depressed RMR in snacking rats. Rather, snacking from weaning causes a shift in gene expression resulting in energy partitioning toward enhanced abdominal fat pad lipogenesis, and adipogenesis and lipid recycling in liver.

Continuous access to snacks from weaning onwards in female rats causes weight gain, insulin insensitivity, and sustained leptin resistance in adulthood.

A large part of the daily intake of children in the U.S. consists of snacks, with the average child consuming three snacks per day. Despite this, little research has been conducted to determine the metabolic and behavioral effects of snacking. Using a developing female rat model, our studies aimed to determine the effects of snacking during development before the protective effects of estrogen on weight gain would be relevant. Additionally, to determine if snack composition is important, we created one healthy and one unhealthy snacking group provided with chow and three snacks each in addition to a chow-only group. We found that both snacking groups experienced increased weight gain, elevated abdominal fat pad mass, prolonged leptin resistance into adulthood, and insulin insensitivity that was not observed in their non-snacking counterparts. These physiological differences were measured despite both snacking groups having a similar caloric intake as the chow-only group throughout the study. In addition to physiological changes, both snacking groups showed a preference for snacks over chow and ate more often during the inactive light phase than typical for rats, with the unhealthy snacking group presenting this behavioral change earlier than the healthy snacking group. Our results suggest that constant access to palatable snacks, which is often the case for children in western countries, alters feeding behaviors in relation to food choice and time of day when eating occurs. Snacking during development seemed to promote signs of metabolic syndrome in adulthood even when excess caloric intake was not observed. Our work further suggests that development is a vulnerable time for palatable snack presentation when prepubertal females lack the protective effects of estrogen and exhibit reduced leptin feedback on food intake. Thus snacking from weaning onward could be a contributor to the current childhood obesity crisis.

Histaminergic regulation of seasonal metabolic rhythms in Siberian hamsters.

We investigated whether histaminergic tone contributes to the seasonal catabolic state in Siberian hamsters by determining the effect of ablation of histaminergic neurons on food intake, metabolic rate and body weight. A ribosomal toxin (saporin) conjugated to orexin-B was infused into the ventral tuberomammillary region of the hypothalamus, since most histaminergic neurons express orexin receptors. This caused not only 75-80% loss of histaminergic neurons in the posterior hypothalamus, but also some loss of other orexin-receptor expressing cells e.g. MCH neurons. In the long-day anabolic state, lesions produced a transient post-surgical decrease in body weight, but the hamsters recovered and maintained constant body weight, whereas weight gradually increased in sham-lesioned hamsters. VO(2) in the dark phase was significantly higher in the lesioned hamsters compared to shams, and locomotor activity also tended to be higher. In a second study in short days, sham-treated hamsters showed the expected seasonal decrease in body weight, but weight remained constant in the lesioned hamsters, as in the long-day study. Lesioned hamsters consumed more during the early dark phase and less during the light phase due to an increase in the frequency of meals during the dark and decreased meal size during the light, and their cumulative food intake in their home cages was greater than in the control hamsters. In summary, ablation of orexin-responsive cells in the posterior hypothalamus blocks the short-day induced decline in body weight by preventing seasonal hypophagia, evidence consistent with the hypothesis that central histaminergic mechanisms contribute to long-term regulation of body weight.

Effects of photoperiod on daily locomotor activity, energy expenditure, and feeding behavior in a seasonal mammal.

The objective of this study was to determine whether the previously observed effects of photoperiod on body weight in Siberian hamsters were due to changes in the daily patterns of locomotor activity, energy expenditure, and/or feeding behavior. Adult males were monitored through a seasonal cycle using an automated comprehensive laboratory animal monitoring system (CLAMS). Exposure to a short-day photoperiod (SD; 8:16-h light-dark cycle) induced a significant decline in body weight, and oxygen consumption (Vo(2)), carbon dioxide production (Vco(2)), and heat production all decreased reaching a nadir by 16 wk of SD. Clear daily rhythms in locomotor activity, Vo(2), and Vco(2) were observed at the start of the study, but these all progressively diminished after prolonged exposure to SD. Rhythms in feeding behavior were also detected initially, reflecting an increase in meal frequency but not duration during the dark phase. This rhythm was lost by 8 wk of SD exposure such that food intake was relatively constant across dark and light phases. After 18 wk in SD, hamsters were transferred to a long-day photoperiod (LD; 16:8-h light-dark cycle), which induced significant weight gain. This was associated with an increase in energy intake within 2 wk, while Vo(2), Vco(2), and heat production all increased back to basal levels. Rhythmicity was reestablished within 4 wk of reexposure to long days. These results demonstrate that photoperiod impacts on body weight via complex changes in locomotor activity, energy expenditure, and feeding behavior, with a striking loss of daily rhythms during SD exposure.

Loss of prokineticin receptor 2 signaling predisposes mice to torpor.

The genes encoding prokineticin 2 polypeptide (Prok2) and its cognate receptor (Prokr2/Gpcr73l1) are widely expressed in both the suprachiasmatic nucleus and its hypothalamic targets, and this signaling pathway has been implicated in the circadian regulation of behavior and physiology. We have previously observed that the targeted null mutation of Prokr2 disrupts circadian coordination of cycles of locomotor activity and thermoregulation. We have now observed spontaneous but sporadic bouts of torpor in the majority of these transgenic mice lacking Prokr2 signaling. During these torpor bouts, which lasted for up to 8 h, body temperature and locomotor activity decreased markedly. Oxygen consumption and carbon dioxide production also decreased, and there was a decrease in respiratory quotient. These spontaneous torpor bouts generally began toward the end of the dark phase or in the early light phase when the mice were maintained on a 12:12-h light-dark cycle and persisted when mice were exposed to continuous darkness. Periods of food deprivation (16-24 h) induced a substantial decrease in body temperature in all mice, but the duration and depth of hypothermia was significantly greater in mice lacking Prokr2 signaling compared with heterozygous and wild-type littermates. Likewise, when tested in metabolic cages, food deprivation produced greater decreases in oxygen consumption and carbon dioxide production in the transgenic mice than controls. We conclude that Prokr2 signaling plays a role in hypothalamic regulation of energy balance, and loss of this pathway results in physiological and behavioral responses normally only detected when mice are in negative energy balance.

Central lipoprivation-induced suppression of luteinizing hormone pulses is mediated by paraventricular catecholaminergic inputs in female rats.

The present study aims to clarify the role of fatty acids in regulating pulsatile LH secretion in rats. To produce an acute central lipoprivic condition, mercaptoacetate (MA), an inhibitor of fatty acids oxidation, was administered into the fourth cerebroventricle (4V) in ad libitum fed ovariectomized (OVX) rats (0.4, 2, and 10 micromol/rat) with or without an estradiol (E2) implant producing diestrus plasma E2 levels. Pulsatile LH secretion was suppressed by 4V MA administration in a dose-dependent manner in both OVX and OVX plus E2 rats. Mean LH levels and LH pulse frequency and amplitude were significantly reduced by the highest dose of MA in OVX rats, and by the middle and highest dose of MA in E2-treated rats, suggesting that estrogen enhanced LH suppression. Blood glucose levels increased immediately after the highest dose of MA in both groups. Fourth ventricular injection of trimetazidine (2 and 3 micromol/rat), another inhibitor of fatty acids oxidation, also inhibited pulsatile LH release, resulting in significant and dose-dependent suppression of LH pulse frequency and an increase in blood glucose levels in OVX plus E2 rats. In contrast, peripheral injection of the highest 4V dose of MA (10 micromol/rat) did not alter LH release or blood glucose levels. Microdialysis of the hypothalamic paraventricular nucleus (PVN) revealed that norepinephrine release in the region was increased by 4V MA administration. Preinjection of alpha-methyl-p-tyrosine, a catecholamine synthesis inhibitor, into the PVN completely blocked the lipoprivic inhibition of LH and the counter-regulatory increase in blood glucose levels in OVX plus E2 rats. Together, these studies indicate that fatty acid availability may be sensed by a central detector, located in the lower brainstem to maintain reproduction, and that noradrenergic inputs to the PVN mediate this lipoprivic-induced suppression of LH release.

Paraventricular alpha1- and alpha2-adrenergic receptors mediate hindbrain lipoprivation-induced suppression of luteinizing hormone pulses in female rats.

Acute central lipoprivation suppresses pulsatile luteinizing hormone (LH) release and increases blood glucose levels through noradrenergic input to the hypothalamic paraventricular nucleus (PVN) in female rats. The present study was conducted to identify adrenergic receptor subtypes involved in central lipoprivation-induced suppression of pulsatile LH secretion and increases in plasma glucose levels in female rats. Acute hindbrain lipoprivation was produced by injection into the fourth cerebroventricle (4V) of 2-mercaptoacetate (MA), an inhibitor of fatty acid oxidation, in estradiol-implanted ovariectomized rats. Two min before MA injection, alpha1-, alpha2- or beta-adrenergic receptor antagonist was injected into the PVN. Injection of MA into the 4V suppresses pulsatile LH release in PVN vehicle-treated rats, whereas pretreatment of animals with injection of alpha1- or alpha2-adrenergic antagonist into the PVN blocked the effect of the 4V MA injection on LH pulses. beta-Adrenergic antagonist did not affect MA-induced suppression of LH pulses. The counter-regulatory increase in plasma glucose levels after 4V MA injection was also partially blocked by pretreatment with alpha1- and alpha2-adrenergic receptor antagonists. These results suggest that alpha1- and alpha2-adrenergic receptors in the PVN mediate hindbrain lipoprivation-induced suppression of LH release and counter-regulatory increases in plasma glucose levels in female rats.

Acute lipoprivation suppresses pulsatile luteinizing hormone secretion without affecting food intake in female rats.

The present study examined the effect of acute lipoprivation on pulsatile luteinizing hormone (LH) secretion in both normal-fat diet, ad libitum-fed and fasted female rats. To produce an acute lipoprivic condition, mercaptoacetate (MA), an inhibitor of fatty acid oxidation, was administered intraperitoneally to ad libitum-fed or 24-h fasted ovariectomized (OVX) rats with or without an estradiol (E2) implant, that produces a negative feedback effect on LH pulses. The steroid treatment was performed to determine the effect of estrogen on lipoprivic changes in LH release, because estrogen enhances fasting- or glucoprivation-induced suppression of LH pulses. Pulsatile LH secretion was suppressed by MA administration in a dose-dependent manner in the ad libitum-fed OVX and OVX+E2 rats. LH pulses were more severely suppressed in the 24-h-fasted OVX and OVX+E2 rats compared to the ad libitum-fed rats. Estrogen slightly enhanced lipoprivic suppression but the effect was not significant. In the present study, increased plasma glucose and free-fatty acid concentrations may indicate a blockade of fatty acid metabolism by the MA treatment, but food intake was not affected by any of the MA doses. Acute vagotomy did not block lipoprivic suppression of LH pulses. Thus, the present study indicates that lipid metabolism is important for maintenance of normal reproductive function even in rats fed a normal-fat diet and lipoprivation may be more critical in fasted animals that probably rely more heavily on fatty acid oxidation to maintain appropriate metabolic fuel levels. In addition, failure of blockade of lipoprivic LH inhibition by vagotomy suggests that lipoprivic information resulting in LH suppression is not transmitted to the brain via the vagus nerve.

Temporal expression of estrogen receptor alpha in the hypothalamus and medulla oblongata during fasting: a role of noradrenergic neurons.

Fasting-induced LH suppression is augmented by estrogen in female rats. We investigated the temporal changes in the number of estrogen receptor alpha (ERalpha)-immunoreactive (ir) cells in various brain regions in ovariectomized rats fasted for 6, 24, 30, and 48 h, commencing at 1300 h. We also determined the anatomical relationship of ERalpha immunoreactivity and dopamine-beta-hydroxylase (DBH) neurons in the A2 region of the nucleus of the solitary tract (NTS) and the paraventricular nucleus (PVN). The number of ERalpha-ir cells significantly increased after 30 h from the onset of fasting in the PVN and NTS compared with the unfasted controls and was sustained until 48 h. In the A2 region of 48-h fasted rats, 46.75% DBH-ir cells expressed ERalpha, and this was significantly higher than in unfasted controls (8.16% DBH-ir cells expressed ERalpha). In the PVN, most ERalpha-ir neurons were juxtaposed with DBH-ir varicosities. These results suggest that ERalpha is expressed in specific brain regions at a defined time from the onset of fasting. In addition, the anatomical relationship of noradrenergic and ERalpha-ir neurons in the A2 region and PVN may suggest a role for estrogen in increasing the activity of noradrenergic neurons in the A2 region and enhancing sensitivity of the PVN to noradrenergic input arising from the lower brainstem and thereby augmenting the suppression of LH secretion during fasting.

Accelerated rehabilitation after total knee replacement.

This study records the length of hospital stay of 50 total knee arthroplasty patients involved in an accelerated postoperative rehabilitation protocol, and a control group of patients undergoing routine rehabilitation. This protocol involved modifications to normal knee replacement procedure, including infiltration of bupivacaine and adrenaline to the divided tissue layers at the time of surgery, spinal anaesthesia, and mobilisation on the day of surgery. These modifications were combined with an organised multidisciplinary approach anticipating issues that may delay discharge. In addition, patients and hospital staff were encouraged to expect an earlier discharge from the hospital. The mean length of hospital stay after surgery was reduced to 3.6 (S.D. 1.0) days, from a previous departmental average of 10.5 days. The control group inpatient stay was 6.6 (S.D. 2.6) days. Plasma bupivacaine levels were found to be well within safe levels, and pain records indicated that the protocol did not cause increased levels of discomfort. American Knee Society and Oxford knee scores demonstrated good levels of knee function at 6 weeks post surgery. In addition, it was noted that no postoperative blood transfusions were required. This is regarded as a significant further benefit.

Fourth ventricular alloxan injection suppresses pulsatile luteinizing hormone release in female rats.

Previous studies have suggested the presence of a glucose-sensing mechanism in the hindbrain that appears to regulate reproductive function as well as feeding behavior. The ependymocytes lining the ventricular wall of the hindbrain showed immunoreactivities to pancreatic glucokinase (GK), a key enzyme for glucose sensing in pancreatic B cells. Our goal in the present study was to test whether the GK-immunopositive ependymocytes in the wall of the fourth cerebroventricle (4V) play a role in regulating gonadal activity. Our approach was to determine the effect of injecting alloxan, a GK inhibitor, into the 4V on pulsatile luteinizing hormone (LH) secretion. Estrogen-primed ovariectomized rats received an injection of alloxan (10 or 20 microg/animal) into the 4V and blood samples were collected every 6 min for 3 h for measurement of blood LH, corticosterone and glucose levels. Pulsatile LH secretion was suppressed after alloxan injection and all pulse parameters were significantly (P<0.05) inhibited by 20 microg alloxan. Plasma corticosterone levels were increased significantly (P<0.05) by 20 microg alloxan, suggesting that LH pulse suppression by alloxan may be at least partly mediated by activation of the hypothalamo-pituitary-adrenal axis. The present results suggest that acute suppression of GK activity in the hindbrain inhibits pulsatile LH secretion in female rats, and supports the idea that GK-immunopositive ependymocytes may sense glucose levels in the cerebrospinal fluid and play a role in regulation of LH secretion.

Involvement of brainstem catecholaminergic inputs to the hypothalamic paraventricular nucleus in estrogen receptor alpha expression in this nucleus during different stress conditions in female rats.

In the present study, we determined the involvement of brainstem catecholaminergic inputs to the paraventricular nucleus (PVN) on estrogen receptor alpha (ERalpha) expression in this nucleus during conditions of 48-h fasting, 2-deoxy-d-glucose (2DG)-induced acute glucoprivation and 1-h immobilization, in ovariectomized rats. Our approach was to examine the effect of lesioning catecholaminergic inputs to the PVN using DSAP [saporin-conjugated anti-DBH (dopamine-beta-hydroxylase)]. Bilateral injection of DSAP into the PVN, 2 wk before stress, prevented fasting-, glucoprivation-, and immobilization-induced increase in ERalpha-immunopositive cells in the PVN. The DBH-immunoreactive (ir) terminals in the PVN were severely depleted by DSAP injection in all experimental groups. Among the brainstem noradreneregic cell groups examined, DBH-ir cell bodies were significantly reduced in the A2 region of all experimental groups treated with DSAP compared with the saporin- and vehicle-injected controls. PVN DSAP injection caused a small, but not significant, decrease in A1 DBH-ir cell bodies in fasted and immobilized rats, and a significant, but slight, reduction in A1 DBH-ir cell bodies of iv 2DG- injected rats compared with PVN vehicle-injected or PVN saporin-injected controls. The A6 DBH-ir cell bodies in all experimental groups treated with DSAP, saporin, or vehicle did not show any significant difference. These results suggest that the brainstem catecholaminergic inputs to the PVN, especially from the A2 cell group, may play a major role in mediating the induction of ERalpha expression in the PVN by metabolic stressors such as fasting, acute glucoprivation, and less specific stressors, such as immobilization, in female rats.

Immunotoxic destruction of distinct catecholaminergic neuron populations disrupts the reproductive response to glucoprivation in female rats.

We tested the hypothesis that hindbrain catecholamine (norepinephrine or epinephrine) neurons, in addition to their essential role in glucoprivic feeding, are responsible for suppressing estrous cycles during chronic glucoprivation. Normally cycling female rats were given bilateral injections of the retrogradely transported ribosomal toxin, saporin, conjugated to monoclonal dopamine beta-hydroxylase antibody (DSAP) into the paraventricular nucleus (PVN) of the hypothalamus to selectively destroy norepinephrine and epinephrine neurons projecting to the PVN. Controls were injected with unconjugated saporin. After recovery, we assessed the lesion effects on estrous cyclicity under basal conditions and found that DSAP did not alter estrous cycle length. Subsequently, we examined effects of chronic 2-deoxy-d-glucose-induced glucoprivation on cycle length. After two normal 4- to 5-d cycles, rats were injected with 2-deoxy-d-glucose (200 mg/kg every 6 h for 72 h) beginning 24 h after detection of estrus. Chronic glucoprivation increased cycle length in seven of eight unconjugated saporin rats but in only one of eight DSAP rats. Immunohistochemical results confirmed loss of dopamine beta-hydroxylase immunoreactivity in PVN. Thus, hindbrain catecholamine neurons with projections to the PVN are required for inhibition of reproductive function during chronic glucose deficit but are not required for normal estrous cyclicity when metabolic fuels are in abundance.

Glucoprivic regulation of estrous cycles in the rat.

Previous research has shown that glucoprivation induced by chronic 2-deoxy-D-glucose (2DG) treatment extends estrous cycle length and disrupts reproductive behaviors in female hamsters, similar to food deprivation. Such treatment also suppresses food intake, which is reversed in male rats by reducing brain histamine levels prior to 2DG treatment. We, therefore, determined if 2DG extends estrous cycles in the female rat and if this is due to elevated brain histamine levels. We measured estrous cycle length during 2DG-induced glucoprivation, in the presence and absence of alpha-fluoromethylhistidine (FMH), a treatment that reduces brain histamine levels. Adult female rats were treated for 72 h with either saline (n = 8), 2DG (200 mg/kg S.C. every 6 h; n = 9), or FMH (100 mg/kg i.p. daily) + 2DG (200 mg/kg; n = 7). An additional group was treated with FMH (100 mg/kg i.p.; n = 5) alone. To determine if 2DG extends estrous cycles due to glucoprivation or to decreased caloric intake, a group of rats (n = 7) received a reduced diet equal to the mean daily food intake of rats receiving 2DG alone. 2DG induced more long estrous cycles compared to rats receiving saline, FMH + 2DG, or FMH alone. In rats treated with FMH + 2DG, the percentage of 4-5-day cycles was similar to that of saline-treated rats, and a high percentage of 4-5-day cycles was also observed in rats receiving a reduced diet. These data suggest that 2DG does not suppress estrous cycles through a decrease in total calorie intake, but rather by inducing glucoprivation. In addition, during 2DG-induced glucoprivation, elevated brain histamine levels contribute to the mechanism that suppresses reproductive function.

Effects of acute inhibition of fatty acid oxidation on latency to seizure and concentrations of beta hydroxybutyrate in plasma of rats maintained on calorie restriction and/or the ketogenic diet.

The present study was designed to evaluate the effects of acute inhibition of fatty acid oxidation on plasma levels of beta hydroxybutyrate and latency to PTZ-induced seizures in ad libitum- (AL), calorie-restricted normal rodent chow- (CR), and calorie-restricted ketogenic diet (KD)-fed young rats. Young (day 23) Sprague-Dawley rats were fasted for 8 h and then fed their respective diets for 21 days. On day 21 of the diet rats in each group received either saline or the fatty acid oxidation inhibitor mercaptoacetate (MA; 46 mg/kg intraperitoneally (i.p.). Two hours later, all rats received pentylenetetrazole (PTZ; 10 mg/kg; i.p.) every 10 min until seizure onset. Results demonstrated that KD-fed rats had the longest (P<0.05) latency to PTZ-induced seizures. KD-fed rats administered an acute dose of MA had lower (P<0.01) levels of beta hydroxybutyrate in plasma and shorter latency to PTZ-induced seizures compared with control KD-fed rats. However, there was not a significant positive correlation (P>0.10) between plasma beta hydroxybutyrate and latency to seizure, suggesting that beta hydroxybutyrate may be indirectly involved in the antiseizure effects of the KD. Fatty acid oxidation inhibition represents an experimental manipulation that may allow for more precise establishment and evaluation of levels of beta hydroxybutyrate in plasma necessary for antiseizure effects of the KD.