Comparative Study of CRH Microinjections into PVN and CeA Nuclei on Food Intake, Ghrelin, Leptin, and Glucose Levels in Acute Stressed Rats

Introduction: Corticotropin-Releasing Hormone (CRH) is involved in stress and energy homeostasis. On the other hand, CRH receptors also exist within the paraventricular nucleus (PVN) and Central Amygdala (CeA) nuclei. The present study compared the effect of CRH microinjections into PVN and CeA on three consecutive hours and cumulative food intake, internal regulatory factors of food intake, such as serum leptin and ghrelin, as well as blood glucose levels in rats under different acute psychological (Social Stress [SS] and Isolation Stress [IS] group) stresses. Methods: Sixty-six male Wistar rats were randomly allocated to 11 groups: Control, Sham, CRH-PVN, CRH-CeA, SS, IS, SS-CRH-PVN, SS-CRH-CeA, IS-CRH-PVN, and IS-CRH-CeA groups. The CRH (2 µg/kg in 0.5 µL saline) was injected into PVN and CeA nuclei in rats under everyday, acute social stress and isolation stress conditions. Results: Acute isolation and social stresses did not affect cumulative food intake. Whereas isolation stress led to changes in both leptin and glucose levels, social stress reduced only glucose levels. Cumulative food intake significantly decreased under acute CRH injection into the CeA and particularly into the PVN. Blood glucose significantly reduced in all the groups receiving CRH into their CeA. Conclusion: The PVN played a more important role compared to CeA on food intake. These nuclei probably employ different mechanisms for their effects on food intake. Besides, it seems that exogenously CRH injection into the PVN probably had a more anorectic effect than naturally activated CRH by stresses. Acute isolation stress had a greater impact than social stress on leptin level and cumulative food intake. Thus, elevated food intake related to leptin compared to ghrelin and glucose levels in the CRH-PVN group under acute social stress.

Fat Mass Regain in Middle-Aged Mice after Sleeve Gastrectomy is divergent from plasma leptin levels

BackgroundSome degree of weight regain is typically observed in human patients who undergo Sleeve Gastrectomy (SG), even if the majority of them do not return to their presurgical body weight. Although the majority of bariatric surgery patients are middle aged, most preclinical models of bariatric surgery utilize juvenile male mice. A long-term characterization of the response of mature, wild type, obese male mice to SG has not been performed.MethodsEight-month old C57bl/6J obese male mice were randomized to undergo SG, sham surgery without caloric restriction (SH) or sham surgery with caloric restriction to match body weight to the SG group (SWM). Body weight, body composition and glucose tolerance were matched at baseline. Mice were followed for 60 days following their respective surgeries.ResultsSG mice had a more pronounced percent weight loss than the SH group in the first post-operative month (p<0.05), along with fat mass loss (p<0.01). By the second post-operative month, the SG group started to regain fat mass, although it continued to be statistically lower than the SH group (p<0.05). Cumulative food intake was significantly lower in the SG group compared to SH group only in the first post-operative week (p<0.05), with both groups having similar cumulative food intake thereafter (p>0.05). SWM group had a significantly lower cumulative food intake throughout the study, except for week 1 (p<0.01). Glucose tolerance was only demonstrably better in the SG group compared to SH group at 8 weeks post-operatively (p<0.01). Plasma leptin was significantly lower in the SG group compared to both SWM and SH groups group by the second post-operative month (p<0.01), in spite of SG’s increasing fat mass accumulation. In the second post-operative month, both FGF-21 and GDF-15 were increased in the SH group compared to the SG and SWM groups (p<0.05), while there was no difference in plasma insulin among the three groups. Heat production was surprisingly higher in the SH group compared to the other two groups (p<0.05), even though brown adipose tissue Peroxisome Proliferator-Activated Receptor Gamma (PPARg) and Cidea mRNA expression were significantly higher in SG and SWM compared to SH (p<0.01). There was no change in BAT UCP-1 mRNA expression among the groups (p>0.05). There was also no change in fecal lipid content among the groups (p>0.05).ConclusionsSG in obese, middle aged male mice leads is accompanied by fat mass regain in the second post-operative month, while plasma leptin levels continue to be significantly lower. This raises the question of whether the observed fat mass regain consists mostly of visceral adipose tissue.

Peripheral Spexin Inhibited Food Intake in Mice

Spexin (SPX, NPQ), a novel endogenous neuropeptide, was firstly identified by bioinformatics. Spexin gene and protein widely distributed in the central nervous system and peripheral tissues, such as the hypothalamus and digestive tract. The role of spexin in appetite regulation in mammalian is still unclear. The present study was designed to investigate the mechanism and effect of peripheral spexin on food intake in mice. During the light period, an intraperitoneal (i.p.) injection of spexin (10 nmol/mouse) significantly inhibited cumulative food intake at 2, 4, and 6 h after treatment in fasted mice. During the dark period, spexin (1 and 10 nmol/mouse, i.p.) significantly suppressed cumulative food intake at 4 and 6 h after treatment in freely feeding mice. The GALR3 antagonist SNAP37889, not GALR2 antagonist, significantly antagonized the inhibitory effect on cumulative food intake (0–6 h) induced by spexin. Spexin significantly reduced the mRNA level of Npy mRNA, not Agrp, Pomc, Cart, Crh, Orexin, or Mch, in the hypothalamus. Spexin (10 nmol/mouse, i.p.) increased the number of c-Fos positive neurons in hypothalamic AHA and SCN, but not in ARC, DMN, LHA, PVN, SON, or VMH. The hypothalamic p-CaMK2 protein expression was upregulated by spexin. This study indicated that acute peripheral injection of spexin inhibited mouse food intake. The anorectic effect may be mediated by GALR3, and inhibiting neuropeptide Y (NPY) via p-CaMK2 and c-Fos in the hypothalamus.

Inhibition of Mitochondrial Fission and iNOS in the Dorsal Vagal Complex Protects from Overeating and Weight Gain

The dorsal vagal complex (DVC) senses changes in insulin levels and controls glucose homeostasis, feeding behaviour and body weight. Three days of high-fat diet (HFD) in rats is sufficient to induce insulin resistance in the DVC and impair its ability to regulate feeding behaviour. HFD-feeding is associated with increased mitochondrial fission in the DVC and fission is regulated by dynamin-related protein 1 (Drp1). Higher Drp1 activity can inhibit insulin signalling, although the exact mechanisms controlling body weight remain elusive. Here we show that Drp1 activation in DVC leads to higher body weight in rats and Drp1 inhibition in HFD-fed rats reduced body weight gain, cumulative food intake and adipose tissue, and prevented insulin resistance. Rats expressing active Drp1 in the DVC had higher levels of inducible nitric oxide synthase (iNOS) and knockdown of iNOS in the DVC of HFD-fed rats led to a reduction in body weight gain, cumulative food intake and adipose tissue, and prevented insulin resistance. In obese insulin-resistant animals, inhibition of mitochondrial fission or DVC iNOS knockdown restored insulin sensitivity and decreased food intake, body weight and fat deposition. Finally, we show that inhibiting mitochondrial fission in DVC astrocytes is sufficient to protect rats from developing HFD-dependent insulin resistance, hyperphagia, body weight gain and fat deposition. Our study uncovers new molecular and cellular targets for brain regulation of whole-body metabolism, which could inform new strategies to combat obesity and diabetes.

OR04-03 Calcitonin Receptor Expressing Neurons in the PVH Regulate Feeding Behavior

The paraventricular nucleus of the hypothalamus (PVH) is a brain region crucial for energy homeostasis. Abnormal PVH development or damage leads to hyperphagic obesity and energy expenditure deficits underscoring the importance of PVH neuronal activity in energy balance control. Application of salmon calcitonin (sCT) to the PVH suppresses feeding and calcitonin receptor (CalcR) is highly expressed in the PVH of rodents suggesting that CalcR-expressing PVH neurons contribute to energy homeostasis. In situ hybridization reveals that many CalcRPVH neurons express melanocortin-4 receptor (MC4R), a receptor required for normal feeding behavior. To investigate the physiologic roles of CalcRPVH neurons, we generated CalcR-2a-Cre knock-in mice to manipulate CalcR-expressing cells. Deletion of MC4R from CalcR expressing cells using Cre-loxP technology resulted in profound obesity in both male and female mice by 16 weeks of age. This weight gain was attributable to hyperphagia, as cumulative food intake of the MC4R deleted mice was significantly greater than the controls and energy expenditure measurements acquired through CLAMS analysis were not significantly different. To determine the brain regions engaged by CalcRPVH neurons, we used anterograde Cre-dependent viral tracing reagents injected into the PVH of CalcR-Cre mice, and found that CalcRPVH neurons project to brain regions implicated in energy balance control, including the nucleus of the solitary tract and the parabrachial nucleus. To assess the acute effects of activating CalcRPVH neurons, we used DREADD technology to chemogenetically activate CalcRPVH neurons. CalcRPVH neuron activation suppressed feeding but had no significant effect on energy expenditure. To determine if the activity of CalcRPVH neurons is required for energy homeostasis, we silenced them using Cre-dependent tetanus toxin virus. Male mice with tetanus toxin silenced CalcRPVH neurons were obese 7 weeks following injection in part due to greater cumulative food intake; CLAMS analysis revealed no differences in energy expenditure. Mice with silenced CalcRPVH neurons as well as mice with CalcR deleted from the PVH had normal anorectic responses to sCT, suggesting sCT-induced anorexia does not require CalcRPVH neurons or CalcR expression in the PVH. Taken together, these findings suggest CalcRPVH neurons are an essential component of feeding and energy homeostatic circuitry.

A Novel Automated System Yields Reproducible Temporal Feeding Patterns in Laboratory Rodents

ABSTRACT Background The impact of temporal feeding patterns remains a major unanswered question in nutritional science. Progress has been hampered by the absence of a reliable method to impose temporal feeding in laboratory rodents, without the confounding influence of food-hoarding behavior. Objective The aim of this study was to develop and validate a reliable method for supplying crushed diets to laboratory rodents in consistent, relevant feeding patterns for prolonged periods. Methods We programmed our experimental feeding station to deliver a standard diet [StD; Atwater Fuel Energy (AFE) 13.9% fat] or high-fat diet (HFD; AFE 45% fat) during nocturnal grazing [providing 1/24th of the total daily food intake (tdF/I) of ad libitum–fed controls every 30 min] and meal-fed (3 × 1-h periods of ad libitum feeding) patterns in male rats (Sprague-Dawley: 4 wk old, 72–119 g) and mice [C57/Bl6J wild-type (WT): 6 mo old, 29–37 g], and ghrelin-null littermates (Ghr−/−; 27–34 g). Results Grazing yielded accurate, consistent feeding events in rats, with an approximately linear rise in nocturnal cumulative food intake [tdF/I (StD): 97.4 ± 1.5% accurate compared with manual measurement; R2 = 0.86; tdF/I (HFD): 99.0 ± 1.4% accurate; R2 = 0.86]. Meal-feeding produced 3 nocturnal meals of equal size and duration in StD-fed rats (tdF/I: 97.4 ± 0.9% accurate; R2 = 0.90), whereas the second meal size increased progressively in HFD-fed rats (44% higher on day 35 than on day 14; P < 0.01). Importantly, cumulative food intake in grazing and meal-fed rats was identical. Similar results were obtained in WT mice except that less restricted grazing induced hyperphagia (compared with meal-fed WT mice; P < 0.05 from day 1). This difference was abolished in Ghr−/− mice, with meal initiation delayed and meal duration enhanced. Neither pattern elevated corticosterone secretion in rats, but meal-feeding aligned ultradian pulses. Conclusions We have established a consistent, measurable, researcher-defined, stress-free method for imposing temporal feeding patterns in rats and mice. This approach will facilitate progress in understanding the physiologic impact of feeding patterns.

Growth in weight and of some tissues in the bullfrog: fitting nonlinear models during the fattening phase

Knowledge of the growth of animals is important so that zootechnical activity can be more accurate and sustainable. The objective of this study was to describe the live weight, development of liver tissue and fat body, leg growth, and cumulative food intake of bullfrogs during the fattening phase using nonlinear models. A total of 2,375 bullfrog froglets with an initial weight of 7.03 ± 0.16 g were housed in five fattening pens (12 m²). Ten samplings were performed at intervals of 14 days to obtain the variables studied. These data were used to estimate the parameters of Gompertz and logistic models as a function of time. The estimated values of weight (Wm) and food intake (FIm) at maturity and time when the growth rate is maximum (t*) were closer to expected values when the logistic model was used. The Wm values for live weight and liver, adipose and leg weights and the FIm value for food intake were 343.7, 15.7, 19.6, 96.03 and 369.3 g, respectively, with t* at 109, 98, 105, 109 and 107 days. Therefore, the logistic model was the best model to estimate the growth and food intake of bullfrogs during the fattening phase.

Rikkunshito, a Japanese Kampo Medicine, Ameliorates Decreased Feeding Behavior via Ghrelin and Serotonin 2B Receptor Signaling in a Novelty Stress Murine Model

We investigated the effects of rikkunshito (RKT), a ghrelin signal enhancer, on the decrease in food intake after exposure to novelty stress in mice. RKT administration (500 mg/kg,per os) improved the decrease in 6 h cumulative food intake. In control mice, the plasma acylated ghrelin levels significantly increased by 24 h fasting. In contrast, the acylated ghrelin levels did not increase by fasting in mice exposed to the novelty stress. RKT administration to the novelty stress mice showed a significant increase in the acylated ghrelin levels compared with that in the distilled-water-treated control mice. Food intake after administering serotonin 2B (5-HT2B) receptor antagonists was evaluated to clarify the role of 5-HT2Breceptor activation in the decrease in feeding behavior after novelty stress. SB215505 and SB204741, 5-HT2Breceptor antagonists, significantly improved the decrease in food intake after exposure to novelty stress. A component of RKT, isoliquiritigenin, prevented the decrease in 6 h cumulative food intake. Isoliquiritigenin showed 5-HT2Breceptor antagonistic activityin vitro.In conclusion, the results suggested that RKT improves the decrease in food intake after novelty stress probably via 5-HT2Breceptor antagonism of isoliquiritigenin contained in RKT.