Peripheral and central administration of a selective neuropeptide Y Y1 receptor antagonist suppresses ethanol intake by C57BL/6J mice

BACKGROUND: Neuropeptide Y (NPY) is a 36-amino acid neuromodulator that is expressed throughout the central nervous system. Recent genetic and pharmacological evidence suggests that the NPY Y1 receptor modulates ethanol intake. To further characterize the role of the Y1 receptor, we examined voluntary ethanol consumption by mice after administration of [(-)-2-[1-(3-chloro-5-isopropyloxycarbonylaminophenyl)ethylamino]-6-[2-(5-ethyl-4-methyl-1,3-tiazol-2-yl)ethyl]-4-morpholinopyridine] (compound A), a novel and selective Y1 receptor antagonist (Y1RA) that acts centrally on brain receptors when administered peripherally. METHODS: C57BL/6J mice were habituated to drinking a 10% (v/v) ethanol solution by using a two-bottle-choice procedure and were then given an intraperitoneal (ip) injection (5 ml/kg) of the Y1RA (0, 25, 50, or 75 mg/kg). In a second study, mice were given intracerebroventricular infusion of the Y1RA (0, 30, or 100 microg). Finally, we determined whether the Y1RA alters open-field locomotor activity, ethanol-induced sedation (3.8 g/kg, ip), or blood ethanol levels. RESULTS: Relative to control treatment, ip injection (50 and 75 mg/kg) and intracerebroventricular infusion (100 microg) of the Y1RA significantly reduced ethanol consumption and food intake without altering water drinking. However, the Y1RA did not alter open-field locomotor activity, ethanol-induced sedation, or blood ethanol levels. CONCLUSIONS: These data indicate that acute blockade of the NPY Y1 receptor with a systemically bioavailable NPY Y1RA reduces voluntary ethanol consumption by C57BL/6J mice. These results are consistent with observations that hypothalamic infusion of NPY increases ethanol drinking by rats.     

Differential mechanisms and development of leptin resistance in A/J versus C57BL/6J mice during diet-induced obesity

Changes in the biological efficacy of leptin were evaluated in obesity-resistant (A/J) and obesity-prone (C57BL/6J) mice at weaning and after consuming a high-fat (HF) diet for 4 and 8 wk. There was no evidence of leptin resistance in either strain at the start of the study, but after 4 and 8 wk on the HF diet, C57BL/6J mice became unresponsive to ip leptin. C57BL/6J mice responded to intracerebroventricular leptin at these time points but developed peripheral resistance to sympathetic stimulation of retroperitoneal white adipose tissue. In contrast, intracerebroventricular leptin was fully effective in A/J mice, reproducing the complete profile of responses observed in weanling mice. A/J mice were also partially responsive to ip leptin at both time points, increasing uncoupling protein 1 mRNA expression in brown adipose tissue and decreasing leptin mRNA in white adipose tissue. The findings indicate that retention of leptin responsiveness is an important component of the ability of A/J mice to mount a robust adaptive thermogenic response and resist diet-induced obesity.     

Age-related changes in hypothalamic-pituitary-adrenal axis activity of male C57BL/6J mice

As there is little known about age-related changes in the hypothalamic-pituitary-adrenal (HPA) axis of mice, we determined the daily patterns of corticosterone secretion every 2 h, together with adrenocorticotropic hormone (ACTH) release and central HPA axis markers in the morning and evening of 3-, 9- and 16-month-old male C57BL/6J mice. We observed that: (i) corticosterone secretion showed a distinct age-related circadian pattern. During the light period this was expressed by relative hypercorticism in 9-month-old mice and relative hypocorticism in 16-month-old mice. ACTH was elevated at 16 months of age; (ii) mineralocorticoid (MR) and glucocorticoid receptor (GR) mRNA expression in the hippocampus was significantly decreased in 9-month-old mice, whereas in 16-month-old mice, expression was similar to young animals. Circadian variation was modest in all age groups; (iii) the parvocellular hypothalamic paraventricular nucleus (PVN) expressed very high vasopressin mRNA, which was subject to circadian variation in 3- and 9-month-old mice. Furthermore, significant levels of MR mRNA were expressed in the PVN. In conclusion, basal HPA axis activity and expression of its central regulatory markers are age-dependent in mice. This suggests that the capacity to adjust to environmental demands is either a function of age, or depends on different dynamics of the HPA axis.     

Counterintuitive effects of double-heterozygous null melanocortin-4 receptor and leptin genes on diet-induced obesity and insulin resistance in C57BL/6J mice

Circulating levels of leptin correlate with food intake and adiposity. A decline in serum leptin associated with calorie restriction instigates behavioral and metabolic adaptation, increasing appetite and conserving energy. Brain melanocortin-4 receptors (Mc4rs) are important mediators of leptin's effects on appetite and energy expenditure. Because subtle changes in function associated with heterozygous null mutations for either the Leptin (Lep-HET) or Mc4r genes (Mc4r-HET) increase adiposity, we tested the hypothesis that combined heterozygous mutations (Dbl-HET) would severely exacerbate diet-induced obesity (DIO) and insulin resistance in C57BL/6J mice. Serum leptin levels were lower as a function of adiposity in heterozygous Leptin mutants (Lep-HET, Dbl-HET) matched with mice homozygous for the wild-type (WT) Lep gene (Mc4r-HET). Evidence for an additive interaction on adiposity in Dbl-HET mice maintained on a low-fat diet was observed at 10 wk of age. Male but not female mice developed DIO and insulin resistance on a high-fat diet. Compared with WT mice, DIO was more severe in Mc4r-HET but not Lep-HET mice, regardless of sex. However, the response of male and female Dbl-HET mice was different, with males being less and females being more responsive relative to Mc4r-HET. Glucose tolerance of Dbl-HET mice was not significantly different from WT mice in either sex. These results show a complex interaction between the Leptin and Mc4r genes that is influenced by age, gender, and diet. Remarkably, while heterozygous Lep mutations initially exacerbate obesity, in situations of severe obesity, reduced leptin levels may act oppositely and have beneficial effects on energy homeostasis.     

Hepatic glucocorticoid binders in mature and senescent C57BL/6J male mice.

Because of prominent age-related changes in the responses to stress and in steroid metabolism and excretion, the effect of age on fractionated liver glucocorticoid-binding proteins was studied in C57BL/6J male mice. When cytosol, pre-labeled with [3H]corticosterone, was chromatographed on Sephadex G-100, 4 peaks were obtained. Peak 1 (excluded), peak 2 (corresponding to plasma transcortin), and peak 3 corresponding to glucocorticoid receptors isolated from nuclei) showed no significant age differences. This finding is consistent with reports that glucocorticoid-mediated induction of hepatic tyrosine aminotransferase is not altered by aging in rodents. However, there was a striking age-related decrease (80%) in peak 4 (apparent MW 29,000). Competition studies imply that peak 4 binds aldosterone, testosterone, progesterone, and corticosterone (delta4-3-keto steroids), but not estradiol or dt a plasma component. Although the function of peak 4 is not identified, the pattern of highest competition with delta4-3-keto steroids suggests that it is a steroid ring "A" reductase.     

Metabolomic profiling of dietary-induced insulin resistance in the high fat-fed C57BL/6J mouse

The predictive ability of metabolic profiling to detect obesity-induced perturbations in metabolism has not been clearly established. Complex aetiologies interacting with environmental factors highlight the need to understand how specific manipulations alter metabolite profiles in this state. The aim of this study was to determine if targeted metabolomic profiling could be employed as a reliable tool to detect dietary-induced insulin resistance in a small subset of experimental animals (n = 10/treatment). Following weaning, male C57BL/6J littermates were randomly divided into two dietary groups: chow and high fat. Following 12 weeks of dietary manipulation, mice were fasted for 5 h prior to serum collection. The resultant high fat-fed animals were obese and insulin resistant as shown by a euglycaemic-hyperinsulinaemic clamp. Sera were analysed by proton nuclear magnetic resonance spectroscopy, and 46 known compounds were identified and quantified. Multivariate analysis by orthogonal partial least squares discriminant analysis, a projection method for class separation, was then used to establish models of each treatment. Models were able to predict class separation between diets with 90% accuracy. Variable importance plots revealed the most important metabolites in this discrimination to include lysine, glycine, citrate, leucine, suberate and acetate. These metabolites are involved in energy metabolism and may be representative of the perturbations taking place with insulin resistance. Results show metabolomics to reliably describe the metabolic effects of insulin resistance in a small subset of samples and are an initial step in establishing metabolomics as a tool to understand the biochemical signature of insulin resistance.     

Chronic neuropeptide Y infusion into the lateral ventricle induces sustained feeding and obesity in mice lacking either Npy1r or Npy5r expression

Neuropeptide Y (NPY) is a powerful orexigenic neurotransmitter. The NPY Y1 and Y5 receptors have been implicated in mediating the appetite-stimulating activity of NPY. To further investigate the importance of these two receptors in NPY-induced hyperphagia after chronic central administration, we used mice lacking either Npy1r or Npy5r expression. NPY infusion into the lateral ventricle of wild-type mice stimulated food intake and induced obesity over a 7-d period. Fat pad weight as well as plasma insulin, leptin, and corticosterone levels were strongly increased in NPY-treated mice. In addition, NPY infusion resulted in a significant decrease in hypothalamic NPY and proopiomelanocortin expression. Interestingly, the lack of either Npy1r or Npy5r expression in knockout mice did not affect such feeding response to chronic NPY infusion. Moreover, the obesity syndrome that developed in these animals was similar to that in wild-type animals. Taken together, these data strongly suggest biological redundancies between Y1 and Y5 receptor signaling in the NPY-mediated control of food intake.     

Failure of dietary quercetin to alter the temporal progression of insulin resistance among tissues of C57BL/6J mice during the development of diet-induced obesity

Aims/hypotheses  High-fat diets produce obesity and glucose intolerance by promoting the development of insulin resistance in peripheral tissues and liver. The present studies sought to identify the initial site(s) where insulin resistance develops using a moderately high-fat diet and to assess whether the bioflavonoid, quercetin, ameliorates progression of this sequence. Methods  Four cohorts of male C57BL/6J mice were placed on diets formulated to be low-fat (10% of energy from fat), high-fat (45% of energy from fat) or high-fat plus 1.2% quercetin (wt/wt). After 3 and 8 weeks, cohorts were evaluated using euglycaemic–hyperinsulinaemic clamps, metabolomic analysis of fatty acylcarnitines and acute in vitro assessments of insulin signalling among tissues. Results  After 3 and 8 weeks, the high-fat diet produced whole-body insulin resistance without altering insulin-dependent glucose uptake in peripheral tissues. The primary defect was impaired suppression of hepatic glucose production by insulin at both times. Quercetin initially exacerbated the effect of high-fat diet by further increasing hepatic insulin resistance, but by 8 weeks insulin resistance and hepatic responsiveness to insulin were similarly compromised in both high-fat groups. The high-fat diet, irrespective of quercetin, increased short-chain fatty acylcarnitines in liver but not in muscle, while reciprocally reducing hepatic long-chain fatty acylcarnitines and increasing them in muscle. Conclusions/interpretation  Failure of insulin to suppress hepatic glucose output is the initial defect that accounts for the insulin resistance that develops after short-term consumption of a high-fat (45% of energy) diet. Hepatic insulin resistance is associated with accumulation of short- and medium-, but not long-chain fatty acylcarnitines. Dietary quercetin does not ameliorate the progression of this sequence. Electronic supplementary material  The online version of this article (doi:) contains supplementary material, which is available to authorised users.     

Murine gamma herpes virus 68 infection promotes fatty liver formation and hepatic insulin resistance in C57BL/6J mice

Purpose  

Murine gamma herpes virus 68 (MHV68) is a naturally occurring mouse pathogen that is homologous to Epstein–Barr virus. This study was designed to determine the correlation between MHV68 infection and lipid accumulation and insulin resistance in livers of C57BL/6J mice, and to explore the underlying mechanisms.     

Acute effects of acamprosate and MPEP on ethanol Drinking-in-the-Dark in male C57BL/6J mice

Background: Recently, a simple procedure in mice, Drinking‐in‐the‐Dark (DID), was hypothesized to have value for medication development for human alcoholism. In DID, mice are offered intermittent, limited access to ethanol over a series of days during the dark phase that results in rapid drinking to intoxication in predisposed genotypes. Methods: We measured the effects of acamprosate or MPEP, metabotropic glutamate 5 receptor (mGluR5) antagonist, on intake of 20% ethanol, plain tap water or 10% sugar water using the DID procedure in male C57BL/6J mice. Results: Acamprosate (100, 200, 300, or 400 mg/kg) dose dependently decreased ethanol drinking with 300 mg/kg reducing ethanol intake by approximately 20% without affecting intake of plain water or 10% sugar water. MPEP (1, 3, 5, 10, 20, or 40 mg/kg) was more potent than acamprosate with 20 mg/kg reducing ethanol intake by approximately 20% and for longer duration without affecting intake of plain water or 10% sugar water. Conclusions: These results support the hypothesis that mGluR5 signaling plays a role in excessive ethanol intake in DID and suggest DID may have value for screening novel compounds that reduce overactive glutamate signaling for potential pharmaceutical treatment of excessive ethanol drinking behavior.     

Development of an alcohol deprivation and escalation effect in C57BL/6J mice

BACKGROUND: Relapse-like drinking has been studied through the expression of the alcohol deprivation effect (ADE), which is measured by a pronounced increase in ethanol preference and consumption after imposed abstinence. No studies have characterized the ADE in C57BL/6J (B6) mice. The present study examined the effects of length and number of deprivations on the expression of the ADE in B6 mice. METHODS: Adult male B6 mice received 24-hour continuous access to ethanol and water for 6 weeks (baseline). Experiment 1 determined the ADE in mice receiving weekly access to 15% ethanol (i.e., exposed 1 day a week and deprived during the other 6 days) for a total of 10 weeks. Experiments 2 and 3 determined the ADE after a single 2-week deprivation period in mice receiving a single concentration of 15% ethanol or multiple concentrations of 7.5, 15, and 30% ethanol, respectively, followed by weekly access to their respective ethanol solutions for 10 weeks. Experiment 4 determined the ADE after a single 2-week deprivation period, followed by daily access to 15% ethanol. Mice never deprived of ethanol (i.e., continuous access) were used as age-matched drinking controls. RESULTS: The ADE was observed after the initial 6-day deprivation period and was profoundly enhanced (i.e., escalation of the ADE) following weekly reexposure to 15% ethanol. Compared with a single concentration of 15% ethanol, concurrent access to multiple ethanol concentrations resulted in a near 2-fold increase in baseline ethanol consumption. Regardless of having access to single or multiple concentrations of ethanol, the ADE was not observed immediately after a 2-week deprivation period. The ADE was observed (although to a lesser magnitude and duration) following weekly reexposure to single or multiple concentrations of ethanol. Alternatively, following a 2-week deprivation period, mice receiving daily access to 15% ethanol showed a significant decrease in ethanol intake and preference (i.e., negative ADE). CONCLUSIONS: Short-term deprivations followed by repeated intermittent (weekly) reexposure to ethanol produces a robust ADE in B6 mice. Increasing the initial deprivation length to 2 weeks produces various opposing effects, including erasure of an initial ADE, diminished expression and magnitude of the ADE following weekly exposure, and complete reversal of the ADE following daily exposure to ethanol.     

Differential changes in islet amyloid polypeptide (amylin) and insulin mRNA expression after high-fat diet-induced insulin resistance in C57BL/6J mice

Islet amyloid, derived from islet amyloid polypeptide (IAPP or amylin), frequently occurs in type 2 diabetes. Availability of this peptide for amyloid formation may be enhanced by increased islet expression of IAPP. In the insulin resistant state, euglycemia is maintained by hypersecretion of insulin. Whether     

Increased expression of hypothalamic leptin receptor and adiponectin accompany resistance to dietary-induced obesity and infertility in female C57BL/6J mice

BACKGROUND: Obesity is strongly associated with female infertility, but the mechanisms underlying this relationship are largely unknown. METHODS: We investigated the effect of increasing dietary fat percentage upon body mass, hypothalamic neuropeptide gene expression, adipose hormone secretion and fertility in females of the inbred mouse strains C57BL/6J and DBA/2J. To assess the effect of obesity independent of dietary influence, we also compared these parameters in wild-type female C57BL/6J mice to those congenic for the obesogenic mutations ob/ob and A(y)/a. RESULTS: After 24 weeks, rather than exhibiting an obese, leptin-resistant phenotype like their female DBA/2J counterparts, wild-type female C57BL/6J mice remained lean, fertile and manifested increased hypothalamic LEPR-B expression. Although both mutant genotypes were associated with obesity and subfertility, ob/ob mice demonstrated significantly increased hypothalamic LEPR-B expression, whereas A(y)/a mice had a significant reduction. Interestingly, wild-type female C57BL/6J mice were noted to manifest significantly higher and lower levels of adiponectin and tissue plasminogen activator inhibitor-1 (tPAI-1), respectively, than weight-matched wild-type female DBA/2J mice. CONCLUSIONS: We conclude that (1) resistance to the obese-infertile phenotype in female C57BL/6J mice is associated with increased hypothalamic leptin receptor expression and alterations in adipokine levels consistent with decreased adipose tissue inflammation and (2) that long-standing hyperleptinemic obesity in mice is associated with a downregulation of the hypothalamic leptin receptor.     

Development of high fat diet-induced obesity and leptin resistance in C57Bl/6J mice

OBJECTIVE: To investigate the development of high fat diet-induced obesity and leptin resistance. DESIGN: Two experiments were carried out in this study. Firstly, we fed the mice with a high- or low-fat diet for up to 19 weeks to examine a progressive development of high fat diet-induced obesity. Secondly, we examined peripheral and central exogenous leptin sensitivity in mice fed high- or low-fat diets for 1, 8 or 19 weeks. SUBJECTS: A total of 168 C57BL/6J mice (3 weeks old) were used in this study. MEASUREMENTS: In the first experiment, we measured the body weight, energy intake, adipose tissue mass, tibia bone length, and plasma leptin in mice fed either a high- or low-fat diet for 1, 8, 15 and 19 weeks. In the second experiment, body weight change and cumulative energy intake were measured at 6 h intervals for 72 h after leptin injection in mice fed a high- or low-fat diet for 1, 8 or 19 weeks. RESULTS: The results from the first experiment suggested that the development of high fat diet-induced obesity in mice could be divided into early, middle and late stages. Compared with the mice fed a low-fat diet, the mice fed a high-fat diet showed a gradually increased body weight (+5.2%), fat storage (epididymal plus perirenal; +6.7%) and plasma leptin (+18%) at 1 week; +11.4%, +68.1%, and +223%, respectively, at 8 weeks; and +30.5%, +141%, and +458%, respectively, at 19 weeks. Energy intake of high fat diet-fed mice was equal to that of low fat diet-fed controls for the first 3 weeks; it fell below control levels over the next 5 week period, but began to increase gradually after 8 weeks of high-fat diet feeding and then increased dramatically from 15 weeks to be 14% higher than that of controls after 19 weeks. The results from our second experiment showed that: (1) after 1 week of feeding, the mice fed a high-fat diet were sensitive to a 2 microg/g (body weight) intraperitoneal (i. p.) injection of leptin, with no differences in body weight change or cumulative energy intake post-injection; (2) after 8 weeks of feeding, the mice fed a high-fat diet were insensitive to 2 microg/g (body weight) i.p. leptin, but were sensitive to a 0.1 microg intracerebroventricular (i.c.v.) injection of leptin; (3) after 19 weeks of feeding, the mice fed a high-fat diet were insensitive to 0. 1 microg i.c.v. leptin, but were sensitive to a high dose of 2 microg i.c.v. leptin. CONCLUSIONS: The present study demonstrated that the development of high fat diet-induced obesity (19 weeks) in C57 B1/6J mice could be divided into three stages: (1) an early stage in response to high-fat diet that mice were sensitive to exogenous leptin; (2) a reduced food intake stage when mice had an increase in leptin production and still retained central leptin sensitivity; and (3) an increased food intake stage, accompanied by a reduction of central leptin sensitivity.     

Hormonal,metabolic and morphologic studies of aged C57BL/6J obese mice

Genetically obese mice (C57BL/6J-ob/ob), fed ad libitum, demonstrated a precipitous increase in the spontaneous death rate after 50 weeks. The first signs of morbidity were a ruffled hair coat and a progressive motor ataxia. Necropsy revealed that obese mice had pale and fatty livers, urolithiasis and grossly distended bladders. Microscopically, the hepatocellular changes observed in all aged obese mice included: a loss of orientation of hepatocytes, an enormous variability in the size of both hepatocytes and their nuclei, and an extensive deposition of both large and small lipid droplets, confirmed by an increase content of triacyl3lycerols. A subacute-to-chronic, multifocal, necrotizing hepatitis was also present. Kidneys from aged obese mice contained hypertrophied glomeruli and increased PAS-stained material. Tubular dilation with compaction of the tubular cells was also seen. There were no significant alterations in the microanatomy or mineralization of femurs from obese mice, yet there was a significant increase in plasma alkaline phosphatase activity. In obese mice at 62–63 weeks of age, hyperglycemia was present even in spite of hyperinsulinemia. Pituitary immunoreactive ACTH and its molar ration to pituitary immunoreactive β-endorphin were also increased in obese mice at this age. Even though the etiology of the decreased lifespan of genetically obese mice remains uncertain, the possibility is discussed that an overall defect in the central nervous system may be involved.     

Inhibition of 5alpha-reduced steroid biosynthesis impedes acquisition of ethanol drinking in male C57BL/6J mice

Background: Allopregnanolone (ALLO) is a physiologically relevant neurosteroid modulator of GABA_A receptors, and it exhibits a psychopharmacological profile that closely resembles the post‐ingestive effects of ethanol. The 5α‐reductase inhibitor finasteride (FIN), which inhibits biosynthesis of ALLO and structurally related neurosteroids, was previously demonstrated to reduce the maintenance of limited‐access ethanol consumption. The primary aim of the current work was to determine whether FIN would reduce the acquisition of drinking in ethanol‐naïve mice. Methods: Male C57BL/6J (B6) mice were acclimated to a reverse light/dark schedule, and were provided ad libitum access to chow and water. Following habituation to vehicle injections (VEH; 20% w/v β‐cyclodextrin; i.p.) administered 22‐hour prior to drinking sessions with water only, mice were divided into 3 treatment groups: vehicle control (VEH), 50 mg/kg FIN (FIN‐50), and 100 mg/kg FIN (FIN‐100). Twenty‐two hours after the first treatment, mice were permitted the inaugural 2‐hour limited access to a 10% v/v ethanol solution (10E) and water. The acquisition of 10E consumption and underlying drinking patterns were assessed during FIN treatment (7 days) and subsequent FIN withdrawal (13 days) phases. Results: FIN dose‐dependently blocked the acquisition of 10E drinking and prevented the development of ethanol preference, thereby suggesting that the GABAergic neurosteroids may be important in the establishment of stable drinking patterns. FIN‐elicited reductions in 10E intake were primarily attributable to selective and marked reductions in bout frequency, as no changes were observed in bout size, duration, or lick rates following FIN treatment. FIN‐treated mice continued to exhibit attenuated ethanol consumption after 2 weeks post‐treatment, despite a full recovery in brain ALLO levels. A second study confirmed the rightward and downward shift in the acquisition of ethanol intake following 7 daily FIN injections. While there were no significant group differences in brain ALLO levels following the seventh day of ethanol drinking, ALLO levels were decreased by 28% in the FIN‐50 group. Conclusions: Although the exact mechanism is unclear, FIN and other pharmacological interventions that modulate the GABAergic system may prove useful in curbing ethanol intake acquisition in at‐risk individuals.