Mice with MCH ablation resist diet-induced obesity through strain-specific mechanisms

Genetics and environment contribute to the development of obesity, in both humans and rodents. However, the potential interaction between genes important in energy balance, strain background, and dietary environment has been only minimally explored. We investigated the effects of genetic ablation of melanin-concentrating hormone (MCH), a neuropeptide with a key role in energy balance, with chow and a high-fat diet (HFD) in two different mouse strains, one obesity-prone (C57BL/6) and the other obesity-resistant (129). Substantial differences were seen in wild-type (WT) animals of different strains. 129 animals had significantly lower levels of spontaneous locomotor activity than C57BL/6; however, 129 mice gained less weight on both chow and HFD. In both strains, deletion of MCH led to attenuated weight gain compared with WT counterparts, an effect secondary to increased energy expenditure. In both strains, feeding a HFD led to further increases in energy expenditure in both WT and MCH-KO mice; however, this increase was more pronounced in 129 mice. In addition, mice lacking MCH have a phenotype of increased locomotor activity, an effect also seen in both strains. The relative increase in activity in MCH(-/-) mice is modest in animals fed chow but increases substantially when animals are placed on HFD. These studies reinforce the important role of MCH in energy homeostasis and indicate that MCH is a plausible target for antiobesity therapy.     

Neither agouti-related protein nor neuropeptide Y is critically required for the regulation of energy homeostasis in mice

Agouti-related protein (AgRP), a neuropeptide abundantly expressed in the arcuate nucleus of the hypothalamus, potently stimulates feeding and body weight gain in rodents. AgRP is believed to exert its effects through the blockade of signaling by alpha-melanocyte-stimulating hormone at central nervous system (CNS) melanocortin-3 receptor (Mc3r) and Mc4r. We generated AgRP-deficient (Agrp(-/-)) mice to examine the physiological role of AgRP. Agrp(-/-) mice are viable and exhibit normal locomotor activity, growth rates, body composition, and food intake. Additionally, Agrp(-/-) mice display normal responses to starvation, diet-induced obesity, and the administration of exogenous leptin or neuropeptide Y (NPY). In situ hybridization failed to detect altered CNS expression levels for proopiomelanocortin, Mc3r, Mc4r, or NPY mRNAs in Agrp(-/-) mice. As AgRP and the orexigenic peptide NPY are coexpressed in neurons of the arcuate nucleus, we generated AgRP and NPY double-knockout (Agrp(-/-);Npy(-/-)) mice to determine whether NPY or AgRP plays a compensatory role in Agrp(-/-) or NPY-deficient (Npy(-/-)) mice, respectively. Similarly to mice deficient in either AgRP or NPY, Agrp(-/-);Npy(-/-) mice suffer no obvious feeding or body weight deficits and maintain a normal response to starvation. Our results demonstrate that neither AgRP nor NPY is a critically required orexigenic factor, suggesting that other pathways capable of regulating energy homeostasis can compensate for the loss of both AgRP and NPY.     

Inactivation of the Rcan2 gene in mice ameliorates the age- and diet-induced obesity by causing a reduction in food intake

Obesity is a serious international health problem that increases the risk of several diet-related chronic diseases. The genetic factors predisposing to obesity are little understood. Rcan2 was originally identified as a thyroid hormone-responsive gene. In the mouse, two splicing variants that harbor distinct tissue-specific expression patterns have been identified: Rcan2-3 is expressed predominately in the brain, whereas Rcan2-1 is expressed in the brain and other tissues such as the heart and skeletal muscle. Here, we show that Rcan2 plays an important role in the development of age- and diet-induced obesity. We found that although the loss of Rcan2 function in mice slowed growth in the first few weeks after birth, it also significantly ameliorated age- and diet-induced obesity in the mice by causing a reduction in food intake rather than increased energy expenditure. Rcan2 expression was most prominent in the ventromedial, dorsomedial and paraventricular hypothalamic nuclei governing energy balance. Fasting and refeeding experiment showed that only Rcan2-3 mRNA expression is up-regulated in the hypothalamus by fasting, and loss of Rcan2 significantly attenuates the hyperphagic response to starvation. Using double-mutant (Lep(ob/ob) Rcan2(-/-)) mice, we were also able to demonstrate that Rcan2 and leptin regulate body weight through different pathways. Our findings indicate that there may be an Rcan2-dependent mechanism which regulates food intake and promotes weight gain through a leptin-independent pathway. This study provides novel information on the control of body weight in mice and should improve our understanding of the mechanisms of obesity in humans.     

Divergent effects of leptin in mice susceptible or resistant to obesity.

Consumption of a high-fat diet decreases hypothalamic neuropeptide Y (NPY) and increases proopiomelanocortin (POMC) and brown adipose uncoupling protein (UCP)-1 mRNA in obesity-resistant SWR/J but not obesity-prone C57Bl/6J mice. Although leptin was elevated in both strains in response to a high-fat diet, its role in the development of diet-induced obesity has remained unclear since insulin and other factors that affect similar tissue targets are altered. Thus, we administered recombinant leptin by subcutaneous infusion to chow-fed mice to mimic the changes in plasma leptin across its broad physiologic range. We observed strain differences in responsiveness to reduced and elevated leptin levels. A reduction in leptin during fasting evoked a greater response in C57Bl/6J mice by decreasing energy expenditure and thyroxin, increasing corticosterone and stimulating food intake and weight gain during refeeding. However, C57Bl/6J mice were less responsive to an increase in leptin in the fed state. Conversely, the leptin-mediated response to fasting was blunted in SWR/J mice, whereas an increase in leptin profoundly reduced food intake and body weight in SWR/J mice fed ad libitum. Sensitivity to fasting in C57Bl/6J mice was associated with higher hypothalamic NPY mRNA and reduced POMC and UCP-1 mRNA expression, while the robust response to high leptin levels in SWR/J mice was associated with suppression of NPY mRNA. These results indicate that differences in leptin responsiveness between strains might occur centrally or peripherally, leading to alteration in the patterns of food intake, thermogenesis and energy storage.     

Genetic control of ATGL-mediated lipolysis modulates adipose triglyceride stores in leptin-deficient mice

Dissecting the genetics of complex traits such as obesity allows the identification of causal genes for disease. Here, we show that the BALB/c mouse strain carries genetic variants that confer resistance to obesity induced by leptin-deficiency or a high-fat diet (HFD). We set out to identify the physiological and genetic bases underlying this phenotype. When compared with C57BL6/J ob/ob mice (B6), BALB/c ob/ob mice exhibited decreased food intake, increased thermogenic capacity, and improved fat catabolism, each of which can potentially modify obesity. Interestingly, analysis of F1 ob/ob (progeny of B6 ob/+ × BALB/c ob+) mice revealed that obesity resistance in BALB/c ob/ob mice principally relied upon improved fat mobilization. This was mechanistically explained by increased adipose triglyceride lipase (ATGL) content in adipocytes, along with increased lipolysis and fatty acid oxidation. We conducted a genome-wide scan and defined a quantitative trait locus (QTL) on chromosome 2. BALB/c alleles on chromosome 2 not only associated with the obesity resistance phenotype but also supported increased ATGL content in adipose tissue. In summary, our study provides evidence that leptin-independent control of adipocyte lipolysis rates directly modifies the balance of macronutrient handling and is sufficient to regulate fat mass in the absence of alterations in food intake and energy expenditure.-Marcelin, G., S-M. Liu, X. Li, G. J. Schwartz, and S. Chua.     

Atherosclerotic lesion formation and triglyceride storage in obese apolipoprotein AI-deficient mice

Obese leptin-deficient (ob/ob) mice have increased levels of high-density lipoprotein (HDL) and a unique lipoprotein referred to as low-density lipoprotein (LDL)/HDL1. When crossed onto an apolipoprotein AI (apoAI)-deficient (-/-) background, ob/ob;apoAI-/- mice accumulate LDL/HDL1 in the absence of traditional HDL. To determine the role of LDL/HDL1 in atherosclerosis, C57BL/6, apoAI-/-, ob/ob and ob/ob;apoAI-/- mice were placed on butterfat diet. After 20 weeks, all four groups had a significant increase in total cholesterol levels. The cholesterol in C57BL/6 mice was carried on very low-density lipoprotein (VLDL) and LDL and, in ob/ob and ob/ob;apoAI-/- mice, on HDL and LDL/HDL1. Atherosclerotic lesion area was similar among C57BL/6, ob/ob and ob/ob;apoAI-/- groups despite their dissimilar lipoprotein profiles. Hepatic triglyceride production and VLDL clearance rates were similar among the four groups. The ob/ob;apoAI-/- group had a significant decrease in liver weight and an increase in white adipose tissue (WAT) weight compared to the ob/ob group. Hepatic scavenger receptor class B type I (SR-BI) levels were decreased in both liver and WAT in ob/ob;apoAI-/- compared to ob/ob mice. Conclusions regarding the atherogenicity of LDL/HDL1 were confounded by the differences in lipoprotein profiles among the four groups. However, our studies provide support for the concept that apoAI and SR-BI assist in the partitioning of lipid from adipose tissue to the liver.     

Rutecarpine ameliorates bodyweight gain through the inhibition of orexigenic neuropeptides NPY and AgRP in mice

Orexigenic neuropeptides NPY and AgRP play major roles in feeding and are closely related to obesity and diabetic metabolic syndrome. This study explored the inhibitory effect of rutecarpine on feeding and obesity in high-fat-diet-induced (C57BL/6) and leptin-deficient (ob/ob) obese mice. Both mice strains developed obesity, but the obesity was inhibited by the reduced food intake resulting from rutecarpine treatment (0.01%, p < 0.01). Blood cholesterol, non-fasting glucose, insulin, and leptin levels were reduced, compared with the control group. Rutecarpine inhibited the expression of NPY and AgRP in the arcuate nucleus (ARC) of the hypothalamus and suppressed the expression of both neuropeptides in N29-4 neuronal cells. These results indicate that rutecarpine ameliorates obesity by inhibiting food intake, which involves inhibited expression of the orexigenic neuropeptides NPY and AgRP.     

Hypothalamic NPY,AGRP, and POMC mRNA responses to leptin and refeeding in mice

Food deprivation (FD) increases hypothalamic neuropeptide Y (NPY) and agouti-related protein (AGRP) mRNA levels and decreases proopiomelanocortin (POMC) mRNA levels; refeeding restores these levels. We determined the time course of changes in hypothalamic NPY, AGRP, and POMC mRNA levels on refeeding after 24 h FD in C57BL mice by in situ hybridization. After 24 h deprivation, mice were refed with either chow or a palatable mash containing no calories or were injected with murine leptin (100 microg) without food. Mice were perfused 2 or 6 h after treatment. Food deprivation increased hypothalamic NPY mRNA (108 +/- 6%) and AGRP mRNA (78 +/- 7%) and decreased hypothalamic POMC mRNA (-15 +/- 1%). Refeeding for 6 h, but not 2 h, was sufficient to reduce (but not restore) NPY mRNA, did not affect AGRP mRNA, and restored POMC mRNA levels to ad libitum control levels. Intake of the noncaloric mash had no effect on mRNA levels, and leptin administration after deprivation (at a dose sufficient to reduce refeeding in FD mice) was not sufficient to affect mRNA levels. These results suggest that gradual postabsorptive events subsequent to refeeding are required for the restoration of peptide mRNA to baseline levels after food deprivation in mice.     

Influence of genetic predisposition to diabetes and obesity on mitochondrial function

Inbred mice with the mutation diabetes C57BL/KsJ db+/db+ and the mutation obese C57BL/6J ob/ob displayed a total liver mitochondrial capacity to oxidize glutamate or succinate which was approximately eight times greater than the capacity of the C57BL/6J +/+ control mice. This increase in oxidation capacity was estimated by multiplying the observed twofold increase in each of the following components: total liver weight, the mitochondrial protein content per gram of liver, and glutamate or succinate respiration activity per milligram of liver mitochondrial protein. No significant difference in liver mitochondrial function and capacity for oxidation was observed between db+/db+ and ob/ob mutants, which indicated that these results may be primarily mediated by the genetic factors responsible for obesity and hyperphagia in these mutants, and not by the genetic traits associated with diabetes. These findings may provide a biochemical foundation in support of the thrifty gene hypothesis.     

Epitope mapping of secreted mouse leptin utilizing peripherally administered synthetic peptides

We have recently reported that intraperitoneal (i.p.) administration of synthetic peptide amides corresponding to amino acids 106-140 of mouse leptin significantly reduced food intake and body weight gain in female C57BL/6J ob/ob mice. These results suggested that leptin activity was localized in domains toward its C-terminus between residues 106-140. In the present study, 14 overlapping peptides encompassing the complete sequence of secreted mouse leptin were synthesized, and their effects on body weight and food intake in female C57BL/6 J ob/ob mice were assessed. When given as seven daily 1-mg i.p. injections, only peptides corresponding to amino acids 106-120, 116-130 and 126-140 caused significant reductions in body weight and food intake. These results confirmed our earlier study and suggest that in contrast to the domain encompassed by amino acids 106-140, the N-terminal of mouse leptin between amino acids 21-105 may not contain functional epitopes that can be utilized as lead compounds in the development of peripherally administered bioactive peptide analogs or nonpeptide mimetics of leptin, which may have potential usefulness in treatment of the energy imbalance associated with obesity.     

Influence of genetic obesity in mice on the lipolytic response of isolated adipocytes to isoproterenol and ACTH-(1-24).

The lipolytic response of isolated adipocytes from genetic obese (C57/BL/64 ob/ob) and lean (C57BL/6J +/?) mice to ACTH-(1-24), isoproterenol and glucagon has been studied. The mean cell idameter of adipocytes form ob/ob mice was approximately twice that of lean controls. The adipocytes from obese mice contained on the average approximately six times the amount of triacylglycerol present in the smaller lean mouse adipocyte. Lipolysis was calculated both on a per cell basis (10(5) cells) and per mu mole of triacylglycerol and when expressed on a cell number basis, the larger adipocytes from obese mice showed an ACTH-(1-24) stimulated glycerol release which was quantitatively similar to that of smaller adipocytes from lean mice. When expressed per mu mole of triacylglycerol, the smaller cells from lean animals appeared to be dramatically more responsive to either isoproterenol or ACTH-(1-24). On either basis, ACTH-(1-24) stimulated glycerol release from obese mouse cells was greater than the isoproterenol response. The obese mouse of adipocyte showed selective loss of response to isoproterenol compared to its lean control.     

Substance P, neurokinin A, vasoactive intestinal polypeptide and gastrin releasing peptide in the intestine and pancreas of spontaneously obese-diabetic mice

The concentrations and contents of immunoreactive substance P (SP), neurokinin A (NKA), vasoactive intestinal polypeptide (VIP) and gastrin releasing peptide (GRP) were measured in acid-ethanol extracts of intestine (duodenum-jejunum-ileum) and pancreas of C57BL/KsJ diabetes-obese (db/db) mice, Aston obese-hyperglycaemic (ob/ob) mice, and their respective lean controls. The intestinal concentration of GRP and pancreatic concentrations of VIP and GRP were 36-57% lower in lean Aston mice than lean C57BL/KsJ mice, indicating the influence of genetic background in control mice. Intestinal concentrations of SP and NKA were reduced by 19-33% in the db/db and ob/ob mutants compared with their lean controls, but the intestinal contents of these peptides were normal or greater than normal due to intestinal hypertrophy of the mutant mice. The intestinal VIP concentration was not altered, but the content was increased by 87% and 25% respectively in db/db and ob/ob mice, whereas the intestinal GRP concentration was reduced by 51% in ob/ob mice. Pancreatic concentrations and contents of NKA, VIP and GRP were similar in lean and db/db C57BL/KsJ mice. However, pancreatic concentrations and contents of VIP and GRP were reduced by 51-55% in ob/ob mice compared with their lean controls. The sensitivity of the present assay did not permit accurate determination of the low pancreatic concentrations of SP. The results suggest that the spontaneous ob/ob and db/db syndromes of obesity and diabetes in mice are associated with reduced intestinal concentrations of SP and NKA. The ob/ob mouse also exhibited reductions of intestinal GRP and pancreatic GRP and VIP concentrations. These changes in regulatory peptides may relate to abnormalities of intestinal and possibly pancreatic function in obese and diabetic mutant mice.     

Effects of a novel Y5 antagonist in obese mice: combination with food restriction or sibutramine

Objective: To further address the function of the Y5 receptor in energy homeostasis, we investigated the effects of a novel spironolactone Y5 antagonist in diet-induced obese (DIO) mice. Methods and Procedures: Male C57BL/6 or Npy5r^−/− mice were adapted to high-fat (HF) diet for 6–10 months and were submitted to three experimental treatments. First, the Y5 antagonist at a dose of 10 or 30 mg/kg was administered for 1 month to DIO C57BL/6 or Npy5r^−/− mice. Second, the Y5 antagonist at 30 mg/kg was administered for 1.5 months to DIO C57BL/6 mice, and insulin sensitivity was evaluated using an insulin tolerance test. After a recovery period, nuclear magnetic resonance measurement was performed to evaluate body composition. Third, DIO mice were treated with the Y5 antagonist alone, or in combination with 10% food restriction, or with another anorectic agent, sibutramine at 10 mg/kg, for 1.5 months. Plasma glucose, insulin, and leptin levels, and adipose tissue weights were quantified. Results: The spironolactone Y5 antagonist significantly reduced body weight in C57BL DIO mice, but not in Npy5r^−/− DIO mice. The Y5 antagonist produced a fat-selective loss of body weight, and ameliorated obesity-associated insulin resistance in DIO mice. In addition, the Y5 antagonist combined with either food restriction or sibutramine tended to produce greater body weight loss, as compared with single treatment. Discussion: These findings demonstrate that the Y5 receptor is an important mediator of energy homeostasis in rodents.     

Loss of FXR protects against diet-induced obesity and accelerates liver carcinogenesis in ob/ob mice

Farnesoid X receptor (FXR) is known to play important regulatory roles in bile acid, lipid, and carbohydrate metabolism. Aged (>12 months old) Fxr(-/-) mice also develop spontaneous liver carcinomas. In this report, we used three mouse models to investigate the role of FXR deficiency in obesity. As compared with low-density lipoprotein receptor (Ldlr) knockout (Ldlr(-/-)) mice, the Ldlr(-/-)Fxr(-/-) double-knockout mice were highly resistant to diet-induced obesity, which was associated with increased expression of genes involved in energy metabolism in the skeletal muscle and brown adipose tissue. Such a striking effect of FXR deficiency on obesity on an Ldlr(-/-) background led us to investigate whether FXR deficiency alone is sufficient to affect obesity. As compared with wild-type mice, Fxr(-/-) mice showed resistance to diet-induced weight gain. Interestingly, only female Fxr(-/-) mice showed significant resistance to diet-induced obesity, which was accompanied by increased energy expenditure in these mice. Finally, we determined the effect of FXR deficiency on obesity in a genetically obese and diabetic mouse model. We generated ob(-/-)Fxr(-/-) mice that were deficient in both Leptin and Fxr. On a chow diet, ob(-/-)Fxr(-/-) mice gained less body weight and had reduced body fat mass as compared with ob/ob mice. In addition, we observed liver carcinomas in 43% of young (<11 months old) Ob(-/-)Fxr(-/-) mice. Together these data indicate that loss of FXR prevents diet-induced or genetic obesity and accelerates liver carcinogenesis under diabetic conditions.     

Effects of oral administration of a synthetic fragment of human growth hormone on lipid metabolism

A small synthetic peptide sequence of human growth hormone (hGH), AOD-9401, has lipolytic and antilipogenic activity similar to that of the intact hormone. Here we report its effect on lipid metabolism in rodent models of obesity and in human adipose tissue to assess its potential as a pharmacological agent for the treatment of human obesity. C57BL/6J (ob/ob) mice were orally treated with either saline (n = 8) or AOD-9401 (n = 10) for 30 days. From day 16 onward, body weight gain in AOD-9401-treated animals was significantly lower than that of saline-treated controls. Food consumption did not differ between the two groups. Analyses of adipose tissue ex vivo revealed that AOD-9401 significantly reduced lipogenic activity and increased lipolytic activity in this tissue. Increased catabolism was also reflected in an acute increase in energy expenditure and glucose and fat oxidation in ob/ob mice treated with AOD-9401. In addition, AOD-9401 increased in vitro lipolytic activity and decreased lipogenic activity in isolated adipose tissue from obese rodents and humans. Together, these findings indicate that oral administration of AOD-9401 alters lipid metabolism in adipose tissue, resulting in a reduction of weight gain in obese animals. The marked lipolytic and antilipogenic actions of AOD-9401 in human adipose tissues suggest that this small synthetic hGH peptide has potential in the treatment of human obesity.     

Obesity and endocrine dysfunction in mice with deletions of both neuropeptide Y and galanin

Neuropeptide Y (NPY) and galanin have both been implicated in the regulation of body weight, yet mice bearing deletions of either of these molecules have unremarkable metabolic phenotypes. To investigate whether galanin and NPY might compensate for one another, we produced mutants lacking both neuropeptides (GAL(-/-)/NPY(-/-)). We found that male GAL(-/-)/NPY(-/-) mice ate significantly more and were much heavier (30%) than wild-type (WT) controls. GAL(-/-)/NPY(-/-) mice responded to a high-fat diet by gaining more weight than WT mice gain, and they were unable to regulate their weight normally after a change in diet. GAL(-/-)/NPY(-/-) mice had elevated levels of leptin, insulin, and glucose, and they lost more weight than WT mice during chronic leptin treatment. Galanin mRNA was increased in the hypothalamus of NPY(-/-) mice, providing evidence of compensatory regulation in single mutants. The disruption of energy balance observed in GAL(-/-)/NPY(-/-) double knockouts is not found in the phenotype of single knockouts of either molecule. The unexpected obesity phenotype may result from the dysregulation of the leptin and insulin systems that normally keep body weight within the homeostatic range.     

Obesity causes very low density lipoprotein clearance defects in low-density lipoprotein receptor-deficient mice

We have reported that obese leptin-deficient mice (ob/ob) lacking the low-density lipoprotein receptor (LDLR(-/-)) develop severe hyperlipidemia and spontaneous atherosclerosis. In the present study, we show that obese leptin receptor-deficient mice (db/db) lacking LDLR have a similar phenotype, even in the presence of elevated plasma leptin levels. We investigated the mechanism for the hyperlipidemia in obese LDLR(-/-) mice by comparing lipoprotein production and clearance rates in C57BL/6, ob/ob, LDLR(-/-) and ob/ob;LDLR(-/-) mice. Hepatic triglyceride production rates were equally increased ( approximately 1.4-fold, P<.05) in both LDLR(-/-) and ob/ob;LDLR(-/-) mice compared to C57BL/6 and ob/ob mice. LDL clearance was decreased ( approximately 1.3- fold, P<.01) to a similar extent in LDLR(-/-) and ob/ob;LDLR(-/-) mice compared to C57BL/6 and ob/ob controls. While VLDL clearance was delayed in LDLR(-/-) compared to C57BL/6 and ob/ob mice (2-fold, P<.001), this delay was exaggerated in ob/ob;LDLR(-/-) mice (3.8-fold, P<001). The VLDL clearance defects were due to decreased hepatic uptake compared to C57BL/6 (54% and 26% for LDLR(-/-) and ob/ob;LDLR(-/-), respectively, P<.001). When VLDL was collected from C57BL/6, ob/ob, LDLR(-/-), and ob/ob;LDLR(-/-) donors and injected into LDLR(-/-) recipient mice, counts remaining in the liver were 1.4-fold elevated in mice receiving LDLR(-/-) VLDL and 2-fold increased in mice receiving ob/ob;LDLR(-/-) VLDL compared to controls receiving C57BL/6 VLDL (P<.01). Thus, the increase in plasma lipoproteins in ob/ob;LDLR(-/-) mice is caused by delayed VLDL clearance. This appears to be due to defects in both the liver and the lipoproteins themselves in these obese mice.     

Effects of estrogen on food intake, body weight, and temperature of male and female obese mice

Estradiol benzoate (EB) treatment of male and female C57BL/6J ob/ob mice for 32 days led to decreased body weight (20%), percentage body fat (8%) and carcass protein content (12%) when compared with non-EB-treated obese control mice. Estradiol reduced the caloric intakes of both genders by 25-35%, but did not affect body temperature regulation. Circulating glucose and insulin concentrations were also lowered by estrogens, although hyperinsulinemia persisted. Since post-treatment body weight changes correlated with daily food intakes (r = 0.81) rather than to rectal temperatures (r = -0.19), it appears that hypophagia provided a greater contribution to the estrogen-mediated reductions of growth and carcass fat than did altered energy expenditure for thermoregulation. While these data show that EB treatment does reduce the severity of some metabolic disturbances in a genetic model of type II diabetes, long-term estrogens do not appear to offer substantial advantages in the treatment of obesity or diabetes when compared with the effects of caloric restriction alone.     

Npy deletion in an alcohol non-preferring rat model elicits differential effects on alcohol consumption and body weight

Neuropeptide Y(NPY) is widely expressed in the central nervous system and influences many physiological processes.It is located within the rat quantitative trait locus(QTL) for alcohol preference on chromosome 4.Alcohol-nonpreferring(NP) rats consume very little alcohol,but have significantly higher NPY expression in the brain than alcohol-preferring(P) rats.We capitalized on this phenotypic difference by creating an Npy knockout(KO) rat using the inbred NP background to evaluate NPY effects on alcohol consumption.Zinc finger nuclease(ZNF) technology was applied,resulting in a 26-bp deletion in the Npy gene.RT-PCR,Western blotting and immunohistochemistry confirmed the absence of Npy mRNA and protein in KO rats.Alcohol consumption was increased in Npy~(+/-) but not Npy~(-/-) rats,while Npy~(-/-) rats displayed significantly lower body weight when compared to Npy~(+/+) rats.In whole brain tissue,expression levels of Npy-related and other alcohol-associated genes,Npy1 r,Npy2r,Npy5 r,Agrp,Mc3 r,Mc4r,Crh and CrMr,were significantly greater in Npy~(-/-) rats,whereas Pome and Crhr2 expressions were highest in Npy~(+/-) rats.These findings suggest that the NPY-system works in close coordination with the melanocortin(MC) and corticotropin-releasing hormone(CRH) systems to modulate alcohol intake and body weight.     

Prolonged fasting induces long-lasting metabolic consequences in mice

To endure prolonged fasting, animals undergo important acute physiological adjustments. However, whether severe fasting also leads to long-term metabolic adaptations is largely unknown. Forty-eight-hour fasting caused a pronounced weight loss in adult C57BL/6 male mice. Seven days of refeeding increased body adiposity to levels above baseline, whereas fasting-induced reductions in lean body mass and energy expenditure were not fully recovered. Respiratory exchange ratio and locomotor activity also remained altered. A fasting/refeeding cycle led to persistent suppression of Pomc mRNA levels and significant changes in the expression of histone deacetylases and DNA methyltransferases in the hypothalamus. Additionally, histone acetylation in the ventromedial nucleus of the hypothalamus was reduced by prolonged fasting and remained suppressed after refeeding. Mice subjected to 48-h fasting 30 days earlier exhibited higher body weight and fat mass compared to aged-matched animals that were never food-deprived. Furthermore, a previous fasting experience altered the changes in body weight, lean mass, energy expenditure and locomotor activity induced by a second cycle of fasting and refeeding. Notably, when acutely exposed to high-palatable/high-fat diet, mice that went through cumulative fasting episodes presented higher calorie intake and reduced energy expenditure and fat oxidation, compared to mice that had never been subjected to fasting. When chronically exposed to high-fat diet, mice that experienced cumulative fasting episodes showed higher gain of body and fat mass and reduced energy expenditure and calorie intake. In summary, cumulative episodes of prolonged fasting lead to hypothalamic epigenetic changes and long-lasting metabolic adaptations in mice.