Repeating of emotional stress prevents development of melanocortin obesity and type 2 diabetes in the mice with the Agouti yellow mutation

Brain melanocortin system (MC-system) participates in regulation of energy homeostasis. Dominant mutation yellow of the Agouti gene leads to the hyperphagia, obesity and type 2 diabetes. Stress is known to inhibit food intake and body weight. The aim of the work was to study effects of repeating emotional stress on food intake and lipid-carbohydrate metabolism in Ay-mice. Male mice of C57B1/6J strain predisposed to the obesity (Ay/a-genotype) and normal (a/a-genotype) were used. In control group food intake, body weight and blood levels of insulin and leptin were increased in Ay/a-mice as compared to a/a-mice. Repeating emotional stress (30 min restraint 3 times a week for 5 weeks) did not alter food intake and indices of lipid-carbohydrate metabolism in a/a-mice and decreased food intake, body weight and blood levels of insulin and leptin in Ay/a-mice. Insulin and leptin blood levels were the same in Ay/a- and a/a-mice on 5 week of treatment. The stress increased basal and stress-induced concentrations of corticosterone to an equal degree in Ay/a- and a/a-mice. Thus, light repeating emotional stress hampered development of obesity and 2 type diabetes in the mice with the Agouti yellow mutation.     

Inhibition of the central melanocortin system decreases brown adipose tissue activity

The melanocortin system is an important regulator of energy balance, and melanocortin 4 receptor (MC4R) deficiency is the most common monogenic cause of obesity. We investigated whether the relationship between melanocortin system activity and energy expenditure (EE) is mediated by brown adipose tissue (BAT) activity. Therefore, female APOE*3-Leiden.CETP transgenic mice were fed a Western-type diet for 4 weeks and infused intracerebroventricularly with the melanocortin 3/4 receptor (MC3/4R) antagonist SHU9119 or vehicle for 2 weeks. SHU9119 increased food intake (+30%) and body fat (+50%) and decreased EE by reduction in fat oxidation (−42%). In addition, SHU9119 impaired the uptake of VLDL-TG by BAT. In line with this, SHU9119 decreased uncoupling protein-1 levels in BAT (−60%) and induced large intracellular lipid droplets, indicative of severely disturbed BAT activity. Finally, SHU9119-treated mice pair-fed to the vehicle-treated group still exhibited these effects, indicating that MC4R inhibition impairs BAT activity independent of food intake. These effects were not specific to the APOE*3-Leiden.CETP background as SHU9119 also inhibited BAT activity in wild-type mice. We conclude that inhibition of central MC3/4R signaling impairs BAT function, which is accompanied by reduced EE, thereby promoting adiposity. We anticipate that activation of MC4R is a promising strategy to combat obesity by increasing BAT activity.     

Ghrelin-induced food intake and growth hormone secretion are altered in melanocortin 3 and 4 receptor knockout mice

Ghrelin stimulates food intake in part by activating hypothalamic neuropeptide Y (NPY) neurons/agouti related peptide (AGRP) neurons. We investigated the role of AGRP/melanocortin signaling in ghrelin-induced food intake by studying melanocortin 3 and 4 receptor knockout (MC3R KO and MC4R KO) mice. We also determined whether reduced ghrelin levels and/or an altered sensitivity to the GH-stimulating effects of ghrelin accompany the obesity syndromes of MC3R KO and MC4R KO mice. Compared to wild-type (WT) mice, the effects of ghrelin on food intake were reduced in MC3R KO and MC4R KO mice and circulating ghrelin levels were reduced in female MC4R KO mice. Female MC3R KO and MC4R KO mice exhibited a diminished responsiveness to the GH-releasing effects of ghrelin. Thus, deletion of the MC3R or MC4R results in a decreased sensitivity to ghrelin and verifies the involvement in the melanocortin system in ghrelin-induced food intake.     

Gamma-MSH/MC3R natriuretic signaling system: The possible explanation for contrasting blood pressure effects of agouti mutation and melanocortin receptor knockout

Overweight and obesity are the major risk factors of arterial hypertension. Recent studies indicate that adipose tissue hormone, leptin, is involved in the development of obesity-induced hypertension. Models of genetically determined obesity in rodents are commonly used to study the pathogenesis of obesity-associated hypertension. One of such models are agouti yellow obese (A^y/a) mice which ubiquitously overexpress agouti protein—an endogenous antagonist of melanocortin receptors normally synthesized only in the hair follicle. In A^y/a mice, agouti protein is synthesized also in the hypothalamus and blocks the anorectic effect of leptin mediated by alpha-melanocyte-stimulating hormone (α-MSH) which binds to melanocortin type 3 and 4 receptors (MC3R and MC4R). Consequently, A^y/a mice are hyperphagic, obese, hyperinsulinemic and hyperleptinemic. Blood pressure is increased in A^y/a mice due to increased serum leptin level. In contrast, blood pressure is reduced in MC4R-null mice despite obesity and hyperleptinemia, and is not increased by the administration of leptin in these animals, suggesting an essential role of the melanocortin pathway in the hypertensive effect of leptin. Herein, I propose the hypothesis which might explain why blood pressure is increased in A^y/a mice but reduced in MC4R−/− mice, although hypothalamic melanocortin signaling is impaired in both models. According to this proposal, in MC4R−/− mice the natriuretic effect of γ-MSH mediated by intrarenal MC3R is preserved and counteracts prohypertensive mechanisms triggered by leptin. In contrast, in A^y/a mice, ubiquitously expressed agouti protein blocks not only hypothalamic MC4R but also renal MC3R and thus impairs γ-MSH-induced natriuresis, leading to blood pressure elevation due to unopposed central and/or peripheral pressor effects of leptin.     

The Yellow Mouse Obesity Syndrome and Mechanisms of Agouti-Induced Obesity

The yellow mouse obesity syndrome is due to dominant mutations at the Agouti locus, which is characterized by obesity, hyperinsulinemia, insulin resistance, hyperglycemia, hyperleptinemia, increased linear growth, and yellow coat color. This syndrome is caused by ectopic expression of Agouti in multiple tissues. Mechanisms of Agouti action in obesity seem to involve, at least in part, competitive melanocortin antagonism. Both central and peripheral effects have been implicated in Agouti-induced obesity. An Agouti-Related Protein (AGRP) has been described recently. It has been shown to be expressed in mice hypothalamus and to act similarly to agouti as a potent antagonist to central melanocortin receptor MC4-R, suggesting that AGRP is an endogenous MC4-R ligand. Mice lacking MC4-R become hyperphagic and develop obesity, implying that agouti may lead to obesity by interfering with MC4-R signaling in the brain and consequently regulating food intake. Furthermore, food intake is inhibited by intracerebro-ventricular injection of a potent melanocortin agonist and was reversed by administration of an MC4-R antagonist. The direct cellular actions of Agouti include stimulation of fatty acid and triglyceride synthesis via a Ca²⁺-dependent mechanism. Agouti and insulin act in an additive manner to increase lipogenesis. This additive effect of agouti and insulin is demonstrated by the necessity of insulin in eliciting weight gain in transgenic mice expressing agouti specifically in adipose tissue. This suggests that agouti expression in adipose tissue combined with hyperinsulinemia may lead to increased adiposity. The roles of melanocortin receptors or agouti-specific receptor(s) in agouti regulation of adipocyte metabolism and other peripheral effects remain to be determined. In conclusion, both central and peripheral actions of agouti contribute to the yellow mouse obesity syndrome and this action is mediated at least in part by antagonism with melanocortin receptors and/or regulation of intracellular calcium.     

Morphological characteristics of liver and white adipose tissue during development of melanocortin obesity in C57Bl/6J Agouti yellow mutant mice

The changes of liver and white adipose tissue (WAT) morphology during development of melanocortin obesity in female Agouti yellow (genotype A Y/a) C57Bl/6J mice have been investigated. Mouse strain of a/a genotype was used as a control. Results have been compared with the hormonal and metabolic changes during development of obesity and type 2 diabete in Agouti yellow mice. The tissues obtained from mice of 8-, 11-, 15- and 22-week old have been analyzed. The morphology of liver and WAT of A(Y) and control animals did not differ during 15 weeks of age. 40% of A(Y) mice revealed liver steatosis (fatty liver) at the age of 22 weeks. In addition, elevation in the inflammatory and proliferating processes in the liver and severe inflammation in WAT has been observed in these animals. Since as early as 15 weeks old A(Y) mice demonstrated the appearance of insulin resistance characteristics we conclude that hormonal and metabolic abnormalities could play a role of the primary factor of pathological reorganization of liver and WAT morphology.     

Mechanisms mediating renal sympathetic activation to leptin in obesity

Leptin plays a critical role in the control of energy homeostasis. The sympathetic cardiovascular actions of leptin have emerged as a potential link between obesity and hypertension. We previously demonstrated that in mice, modest obesity induced by 10 wk of a high-fat diet is associated with preservation of leptin ability to increase renal sympathetic nerve activity (SNA) despite the resistance to the metabolic effects of leptin. Here, we examined whether selective leptin resistance exists in mice with late-stage diet-induced obesity (DIO) produced by 20 wk of a high-fat diet. The decrease in food intake and body weight induced by intraperitoneal or intracerebroventricular injection of leptin was significantly attenuated in the DIO mice. Regional SNA responses to intravenous leptin were also attenuated in DIO mice. In contrast, intracerebroventricularly administered leptin caused contrasting effects on regional SNA in DIO mice. Renal SNA response to intracerebroventricular leptin was preserved, whereas lumbar and brown adipose tissue SNA responses were attenuated. Intact renal SNA response to leptin combined with the increased cerebrospinal fluid leptin levels in DIO mice represents a potential mechanism for the adverse cardiovascular consequences of obesity. Lastly, we examined the role of phosphoinositol-3 kinase (PI3K) and melanocortin receptors (MCR) in mediating the preserved renal SNA response to leptin in obesity. Presence of PI3K inhibitor (LY294002) or MC3/4R antagonist (SHU9119) significantly attenuated the renal SNA response to leptin in DIO and agouti obese mice. Our results demonstrate the importance of PI3K and melanocortin receptors in the transduction of leptin-induced renal sympathetic activation in obesity.     

Increased hypothalamic melanin concentrating hormone gene expression during energy restriction involves a melanocortin-independent, estrogen-sensitive mechanism

Increased expression of melanin concentrating hormone (MCH), an orexigenic neuropeptide produced by neurons in the lateral hypothalamic area (LHA), is implicated in the effect of energy restriction to increase food intake. Since melanocortins inhibit Mch gene expression, this effect of energy restriction to increase Mch signaling may involve reduced hypothalamic melanocortin signaling. Consistent with this hypothesis, we detected increased hypothalamic Mch mRNA levels in agouti (Ay) mice (by 102%; P < 0.05), a model of genetic obesity resulting from impaired melanocortin signaling, compared to wild-type controls. If reduced melanocortin signaling mediates the effect of energy restriction, hypothalamic Mch gene expression in Ay mice should not be increased further by energy restriction, since melanocortin signaling is impaired in these animals regardless of nutritional state. We therefore investigated the effects of energy restriction on hypothalamic Mch gene expression in both Ay mice and in wild-type mice with diet-induced obesity (DIO). Responses in these mice were compared to those induced by administration of 17beta-estradiol (E2) at a dose previously shown to reduce food intake and Mch expression in rats. In both Ay and DIO mice, energy restriction increased hypothalamic Mch mRNA levels (P < 0.05 for each) via a mechanism that was fully blocked by E2. However, E2 did not lower levels of Mch mRNA below basal values in Ay mice, whereas it did so in DIO mice. Thus, the effect of energy restriction to increase hypothalamic Mch gene expression involves an E2-sensitive mechanism that is not altered by impaired melanocortin signaling. By comparison, impaired melanocortin signaling increases hypothalamic Mch gene expression via a mechanism that is insensitive to E2. These findings suggest that while both energy restriction and reduced melanocortin signaling stimulate hypothalamic Mch gene expression, they do so via distinct mechanisms.     

Profound obesity secondary to hyperphagia in mice lacking kinase suppressor of ras 2

The kinase suppressor of ras 2 (KSR2) gene resides at human chromosome 12q24, a region linked to obesity and type 2 diabetes (T2D). While knocking out and phenotypically screening mouse orthologs of thousands of druggable human genes, we found KSR2 knockout (KSR2(-/-)) mice to be more obese and glucose intolerant than melanocortin 4 receptor(-/-) (MC4R(-/-)) mice. The obesity and T2D of KSR2(-/-) mice resulted from hyperphagia which was unresponsive to leptin and did not originate downstream of MC4R. The kinases AMP-activated protein kinase (AMPK) and mammalian target of rapamycin (mTOR) are each linked to food intake regulation, but only mTOR had increased activity in KSR2(-/-) mouse brain, and the ability of rapamycin to inhibit food intake in KSR2(-/-) mice further implicated mTOR in this process. The metabolic phenotype of KSR2 heterozygous (KSR2(+/minus;)) and KSR2(-/-) mice suggests that human KSR2 variants may contribute to a similar phenotype linked to human chromosome 12q24.     

Regulation of lean mass, bone mass, and exercise tolerance by the central melanocortin system

Signaling via the type 4-melanocortin receptor (MC4R) is an important determinant of body weight in mice and humans, where loss of function mutations lead to significant obesity. Humans with mutations in the MC4R experience an increase in lean mass. However, the simultaneous accrual of fat mass in such individuals may contribute to this effect via mechanical loading. We therefore examined the relationship of fat mass and lean mass in mice lacking the type-4 melanocortin receptor (MC4RKO). We demonstrate that MC4RKO mice display increased lean body mass. Further, this is not dependent on changes in adipose mass, as MC4RKO mice possess more lean body mass than diet-induced obese (DIO) wild type mice with equivalent fat mass. To examine potential sources of the increased lean mass in MC4RKO mice, bone mass and strength were examined in MC4RKO mice. Both parameters increase with age in MC4RKO mice, which likely contributes to increases in lean body mass. We functionally characterized the increased lean mass in MC4RKO mice by examining their capacity for treadmill running. MC4R deficiency results in a decrease in exercise performance. No changes in the ratio of oxidative to glycolytic fibers were seen, however MC4RKO mice demonstrate a significantly reduced heart rate, which may underlie their impaired exercise performance. The reduced exercise capacity we report in the MC4RKO mouse has potential clinical ramifications, as efforts to control body weight in humans with melanocortin deficiency may be ineffective due to poor tolerance for physical activity.     

Effects of Pravastatin on Obesity,Diabetes, and Adiponectin in Diet‐induced Obese Mice

Objective: The aim of this study was to investigate the in vivo effects of pravastatin on the development of obesity and diabetes in diet‐induced obese (DIO) mice. Methods and Procedures: We examined food intake, body‐weight changes, visceral white adipose tissue (WAT) adiponectin and resistin levels, and energy metabolism. Results: Treatment with 100 mg/kg/day pravastatin for 28 days decreased diet‐induced weight gain and visceral adiposity. In addition, the weight of the WAT, the triglyceride (TG) contents of the liver and muscles, and the levels of serum insulin improved in the pravastatin‐treated DIO mice. Furthermore, pravastatin treatment changed the WAT adiponectin and resistin mRNA expression and serum levels compared with the controls. Finally, pravastatin treatment increased oxygen consumption and decreased the respiratory quotient (RQ). Discussion: Pravastatin treatment prevents the development of obesity and diabetes in DIO mice. The prevention of obesity may be mediated by increased oxygen consumption and a decrease in the RQ. These results provide novel insights into the use of pravastatin as a therapeutic tool for metabolic syndromes.     

Characterisation of electrogenic nutrient absorption in the <Emphasis Type="Italic">Cftr</Emphasis><Superscript><Emphasis Type="Italic">TgH(neoim)Hgu</Emphasis></Superscript> mouse model

Most cystic fibrosis (CF) patients show an exocrine pancreatic insufficiency that results in lower enzyme and bicarbonate secretion. To test whether an altered function of nutrient transporters might additionally attribute to the lower bodyweight of CF patients we investigated electrogenic absorption of alanine, glycyl-glutamine, glucose and the effect of pH on nutrient absorption by Ussing chambers in a CF mouse model carrying the Cftr TgH(neoim)Hgu mutation. The electrogenic transport of all three nutrients was similar between the D2.129P2(CF/3)-Cftr TgH(neoim)Hgu congenic strain and DBA/2J mice as well as between the B6.129P2(CF/3)-Cftr TgH(neoim)Hgu congenic strain and C57BL/6J mice. This indicates that the Cftr TgH(neoim)Hgu mutation does not affect the electrogenic absorption of alanine, glycyl-glutamine and glucose. In contrast, electrogenic nutrient absorption was reduced in the CF/1-Cftr TgH(neoim)Hgu and CF/3-Cftr TgH(neoim)Hgu inbred strains compared to the HsdOla:MF1, D2.129P2(CF/3)-Cftr TgH(neoim)Hgu and B6.129P2(CF/3)-Cftr TgH(neoim)Hgu strains, whereas no difference was found among the wild-type strains. This indicates that not the Cftr TgH(neoim)Hgu mutation but differences in the genetic background of the CF/1-Cftr TgH(neoim)Hgu and CF/3-Cftr TgH(neoim)Hgu strains compared to HsdOla:MF1, D2.129P2(CF/3)-Cftr TgH(neoim)Hgu and B6.129P2(CF/3)-Cftr TgH(neoim)Hgu strains are associated with the differences in electrogenic nutrient absorption. The electrogenic absorption of alanine, glycyl-glutamine and glucose was not influenced by an acidic pH (5.4) compared to absorption at pH 7.4.     

Effects of low‐level 50 Hz magnetic fields on the level of host defense and on spleen colony formation

The results of 3 sets of experiments on the effects of 22 μT sinusoidal 50 Hz magnetic fields (MF), applied for 1 h on 5 successive days (1 h/5 days), on the level of host defense and on spleen colony formation are reported. The first set of experiments shows the effects on the number of colony‐forming units (CFUs) on the spleen and on the cellularity of the thymus in mice. The MF exposures resulted in an increase in CFUs which was statistically significant with respect to the controls, but not with respect to the shams. Statistically significant changes in the thymic weight and thymic index with respect to both the controls and the shams were measured 1 h after the last MF exposure. In the second set of experiments, the mice were given a sublethal dose of X‐rays (6 Gy), which was followed by exposure 2 h later to the MF. The MF exposure was repeated at the same time of day for 5 days. The number of colonies per spleen showed a consistent, statistically significant increase with MF exposure and the number of CFUs per femur was decreased. In the third set of experiments, bone marrow was taken from mice which had been exposed to 22 μT fields and injected into mice which had been exposed to a lethal dose of X‐rays (9 Gy). The number of CFUs per femur in the recipient mice was shown to be reduced by a statistically significant amount at 1 and 4 days after injection. Bioelectromagnetics 20:57–63, 1999. © 1999 Wiley‐Liss, Inc.     

Voluntary exercise delays monogenetic obesity and overcomes reproductive dysfunction of the melanocortin-4 receptor knockout mouse

The melanocortin system is involved in hypothalamic regulation of energy homeostasis. The melanocortin-4 receptor (MC4R) has been linked to both obesity and reproductive dysfunction. Deletion of the MC4R from the mouse genome has resulted in phenotypes including adult onset obesity, hyperphagia, and difficulty in reproducing when homozygote parents are bred. Additionally, polymorphisms of the human MC4R have been identified in morbidly obese children and adults. Herein, we have identified that voluntary exercise, provided via the presence of a running wheel, impedes the monogenetic obesity (at 20 weeks of age running wheel housed body weight=31+/-1.8 g versus conventionally housed body weight=41+/-2.3 g, a 25% decrease in body weight p<0.01), hyperphagia (average cumulative food intake is not statistically different than wild type mice housed in running wheel cages), and reproductive dysfunction phenotypes associated with the MC4R knockout mice housed by conventional means. These data demonstrate the novel finding that voluntary exercise at a young age may hinder genetically induced obesity.     

Inhibition of pentylenetetrazole-induced seizure in mice by using a 4 Hz magnetic field: a comparative study with a 60 Hz magnetic field

We investigated the comparative effects of 4 and 60 Hz magnetic fields on pentylenetetrazole (PTZ)-induced seizure in mice. For this study, we measured the latent time to seizure, seizure duration, and lethality induced by PTZ in mice exposed to 4 and 60 Hz magnetic fields (MF) for 30 min. Compared to sham-exposed controls, the latent time to tail twitching and seizure in the 4 Hz MF group was significantly decreased while the latent time to seizure in the 60 Hz MF group was significantly increased. The seizure duration in the 4 Hz MF group was significantly decreased while that in the 60 Hz MF group was significantly increased. More importantly, while the mice exposed to a 60 Hz MF experienced significantly increased lethality after seizure convulsion, those exposed to a 4 Hz MF showed no lethality, with a shortening of the duration of seizure. This beneficial effect of a 4 Hz MF on seizure has the same implication as the anti-oxidative effects of a 4 Hz MF observed in our previous work. The results of our current and previous works indicate that a 4 Hz MF may be used as a therapeutic physical agent for the treatment of oxidative stress-induced diseases, including seizure, with or without chemical drugs.     

Food intake reductions and increases in energetic responses by hindbrain leptin and melanotan II are enhanced in mice with POMC-specific PTP1B deficiency

Leptin regulates energy balance through central circuits that control food intake and energy expenditure, including proopiomelanocortin (POMC) neurons. POMC neuron-specific deletion of protein tyrosine phosphatase 1B (PTP1B) (Ptpn1(loxP/loxP) POMC-Cre), a negative regulator of CNS leptin signaling, results in resistance to diet-induced obesity and improved peripheral leptin sensitivity in mice, thus establishing PTP1B as an important component of POMC neuron regulation of energy balance. POMC neurons are expressed in the pituitary, the arcuate nucleus of the hypothalamus (ARH), and the nucleus of the solitary tract (NTS) in the hindbrain, and it is unknown how each population might contribute to the phenotype of POMC-Ptp1b(-/-) mice. It is also unknown whether improved leptin sensitivity in POMC-Ptp1b(-/-) mice involves altered melanocortin receptor signaling. Therefore, we examined the effects of hindbrain administration (4th ventricle) of leptin (1.5, 3, and 6 μg) or the melanocortin 3/4R agonist melanotan II (0.1 and 0.2 nmol) in POMC-Ptp1b(-/-) (KO) and control PTP1B(fl/fl) (WT) mice on food intake, body weight, spontaneous physical activity (SPA), and core temperature (T(C)). The results show that KO mice were hypersensitive to hindbrain leptin- and MTII-induced food intake and body weight suppression and SPA compared with WT mice. Greater increases in leptin- but not MTII-induced T(C) were also observed in KO vs. WT animals. In addition, KO mice displayed elevated hindbrain and hypothalamic MC4R mRNA expression. These studies are the first to show that hindbrain administration of leptin or a melanocortin receptor agonist alters energy balance in mice likely via participation of hindbrain POMC neurons.     

Chronic exposure to an extremely low‐frequency magnetic field induces depression‐like behavior and corticosterone secretion without enhancement of the hypothalamic–pituitary–adrenal axis in mice

An extremely low‐frequency magnetic field (ELF‐MF) is generated by power lines and household electrical devices. Many studies have suggested an association between chronic ELF‐MF exposure and anxiety and/or depression. The mechanism of these effects is assumed to be a stress response induced by ELF‐MF exposure. However, this mechanism remains controversial. In the present study, we investigated whether chronic ELF‐MF exposure (intensity, 3 mT; total exposure, 200 h) affected emotional behavior and corticosterone synthesis in mice. ELF‐MF‐treated mice showed a significant increase in total immobility time in a forced swim test and showed latency to enter the light box in a light–dark transition test, compared with sham‐treated (control) mice. Corticosterone secretion was significantly high in the ELF‐MF‐exposed mice; however, no changes were observed in the amount of the adrenocorticotropic hormone and the expression of genes related to stress response. Quantification of the mRNA levels of adrenal corticosteroid synthesis enzymes revealed a significant reduction in Cyp17a1 mRNA in the ELF‐MF‐exposed mice. Our findings suggest the possibility that high intensity and chronic exposure to ELF‐MF induces an increase in corticosterone secretion, along with depression‐ and/or anxiety‐like behavior, without enhancement of the hypothalamic–pituitary–adrenal axis. Bioelectromagnetics 34:43–51, 2013. © 2012 Wiley Periodicals, Inc.