POMC Expression Research Articles

Omega-3 offers better hypothalamus protection by decreasing POMC expression and elevating ghrelin hormone: a prospective trial to overcome methotrexate-induced anorexia.

Methotrexate (MTX) therapy is widely used in treatment of different types of diseases including inflammatory diseases, autoimmune disorders, and cancer. However, most of patients respond well to MTX, they suffer from multiple side effects including severe anorexia. Omega-3 fatty acid possesses many beneficial biological activities. Therefore, the objective of our study is to explore the effect of the combined modality of omega-3 (400 mg/kg/day) in MTX-induced anorexia in rats. The effect of MTX alone and in combination with omega-3 on the body weight, ghrelin hormone level, histopathological findings of taste buds and hypothalamus and POMC gene expression were investigated. Interestingly, the capability of omega-3 to overcome the anorexic effect of MTX could be manifested by controlling weight loss, increasing serum HDL, elevating the ghrelin level as well as reducing both lesions within taste buds and hypothalamus and hypothalamic POMC gene expression. our findings revealed that the omega-3 might be used as a complementary supplement during the MTX therapy to ameliorate its anorexic effect.

Intermittent fasting, adipokines, insulin sensitivity, and hypothalamic neuropeptides in a dietary overload with high-fat or high-fructose diet in mice

The intermittent fasting (IF) might have benefits on metabolism and food intake. Twelve-week old C57BL/6 J mice were fed a control diet (C, 10% kcal fat), a high-fat diet (HF, 50% kcal fat) or a high-fructose diet (HFru, 50% kcal fructose) for 8 weeks, then half of the animals in each group underwent IF (24 h fed, 24 h fasting) for an additional 4 weeks. Although food intake on the fed day remained the same for all groups, all fasting groups showed a reduction in body mass compared to their counterparts. IF reduced total cholesterol, triacylglycerol, fasting glucose, fasting insulin resistance index, and plasma leptin, but increased plasma adiponectin. IF reduced Leptin gene expression in the HF-IF group, but increased proinflammatory markers in the hypothalamus, also in the C-IF group. Both groups HFru-IF and C-IF, showed alterations in the leptin signaling pathway (Leptin, OBRb, and SOCS3), mainly in the HFru-IF group, suggesting leptin resistance. NPY and POMC neuropeptides labeled the neurons of the hypothalamus by immunofluorescence, corroborating qualitatively other quantitative findings of the study. In conclusion, current results are convincing in demonstrating the IF effect on central regulation of food intake control, as shown by NPY and POMC neuropeptide expressions, resulting in a lower weight gain. Besides, IF improves glycemia, lipid metabolism, and consequently insulin and leptin resistance. However, there is increased expression of inflammatory markers in mouse hypothalamus challenged by the HF and HFru diets, which in the long term may induce adverse effects.

SAT-596 POMC Expression in GABAergic Neurons Suppresses NPY Overexpression and Restores Food Intake in Obese Mice

Hypothalamic arcuate proopiomelanocortin (Arc-POMC) neurons are involved in different physiological processes such as the regulation of energy balance, glucose homeostasis and stress induced analgesia. Since these neurons heterogeneously express different biological markers and project to many hypothalamic and extrahypothalamic areas, it is proposed that Arc-POMC neurons could be classified into different subpopulations having diverse physiological roles. The aim of the present study was to characterize the contribution of the subpopulation of Arc-POMC neurons co-secreting gamma-aminobutyric acid (GABA) neurotransmitter in the control of energy balance. Arc-Pomc expression restricted to GABAergic-POMC neurons was achieved by crossing a reversible Pomc-deficient mouse line (arcPomc-) with a tamoxifen-inducible Gad2-CreER transgenic line. Tamoxifen treatment of arcPomc-/-:Gad2-CreER mice at P60 resulted in Pomc expression in ~25 % of Arc-POMC neurons and ~23 % of Pomc mRNA levels, compared to Gad2-CreER control mice. Rescued mice normalized food intake, glycemia and fasting-induced hyperphagia, and significantly reduced body weight. Energy balance was also improved in arcPomc-/-:Gad2-CreER mice treated with tamoxifen at P25. Distribution analysis of rescued POMC immunoreactive fibers revealed that the DMH is a major target site of GABAergic-POMC neurons. Interestingly, the expression of the orexigenic neuropeptide Y (NPY) in the DMH was increased in arcPomc-/- obese mice but completely restored after Pomc rescue in arcPomc-/-:Gad2-CreER mice. Finally, we performed stereotactic intracerebral injections of fluorescent retrobeads into the DMH followed by in situ hybridization for Gad1 and found that ~75 % of Arc-POMC neurons projecting to the DMH are GABAergic. In conclusion, in the present study we show that the expression of Pomc in the subpopulation of Arc-GABAergic-POMC neurons is sufficient to maintain normal food intake. In addition, we found that DMH-NPY expression is negatively correlated with Pomc expression in GABAergic-POMC neurons, suggesting that food intake may be regulated by an Arc-GABAergic-POMC --> DMH-NPY pathway.

OR16-03 Metabolic Effects Of Hypothalamic Pomc Neurons Generated Postnatally From Tanycytes On A Pomc Null Genetic Background

Hypothalamic proopiomelanocortin (POMC) neurons are an integral part of the central melanocortin system and regulate feeding and energy balance in vertebrates. Tanycytes are radial glial-like cells lining the third ventricle that contain a subpopulation of adult stem cells, which can differentiate under specific circumstances into glia and neurons, including POMC neurons. However, the capacity of these stem cell-derived neurons to fully mature and integrate into existing neural circuits of physiological relevance is unknown. This study systematically tested whether Pomc mRNA-positive cells newly generated from tanycyte precursors can differentiate into melanocortin-secreting POMC neurons, integrate into the normal anatomical projection pathways of these cells and rescue the obesity phenotype caused by the loss of Pomc expression in ArcPomcfneo/fneo mice. We generated an inducible compound genetic mouse model by crossing RaxCreERT2 with the Cre-dependent ArcPomcfneo/fneo and LSL-syp-tdTomato alleles. Rax is expressed exclusively in postnatal tanycytes, thereby limiting tamoxifen-induced recombination of the two floxed alleles by CreERT2 to tanycytes. As expected, tamoxifen treatment of the mice at age 4–5 wk recapitulated endogenous Rax expression 16 wk later as observed by red fluorescent tdTomato expression in all tanycytes. In addition, Cre recombinase-mediated deletion of the floxed-neomycin cassette from the neuronal enhancer region of the ArcPomcfneo alleles relieved their constitutive transcriptional silencing. Consequently, tamoxifen treatment consistently generated a significant number of newly generated POMC neurons from tanycytes (~10% of the POMC neurons in a WT mouse), identified by Pomc FISH and POMC/α-MSH immunofluorescence in the soma and established terminal projections to hypothalamic nuclei including the PVH and DMH involved in energy homeostasis. A subpopulation of these neurons also expressed the synaptophysin-tDTomato reporter. We performed serial body weight, food intake, body composition, oral GTT and insulin measurements with the RaxCreERT2/+, ArcPomcfneo/fneo mice and found no significant differences in any of these metabolic variables compared to untreated obese ArcPomcfneo/fneo mice. These data are consistent with previous studies from our lab suggesting that Pomc expression has to be at least ~30% of normal to mitigate the obesity phenotype in Pomc-null mice. In conclusion, we demonstrated that tanycytes are capable of generating mature Pomc-expressing neurons in the hypothalamus of adult mice. However, we propose that determining the underlying mechanisms involved in the generation of hypothalamic POMC neurons from tanycytes and interventions to increase their number, might lead to a novel approach to treat obesity. Nothing to Disclose: SG, GW, RML, MJL

SAT-308 Effect of Silibinin on ACTH Synthesis and Secretion in Human Adenomatous Corticotropes in Vitro

Silibinin, a milk thistle extract with known hepatoprotective effects, has recently been shown to act upon tumoral corticotropes and revert the Cushingoid phenotype in an allograft mouse model (Riebold et al 2015). Silbinin is known to inhibit HSP90 -a chaperone to the glucocorticoid receptor- thereby restoring sensitivity to glucocorticoid negative feedback in tumoral corticotropes. Aim of the present study was to assess the effect of silibinin on ACTH synthesis and secretion by human corticotrope adenomas in vitro. Methods: Eight human ACTH-secreting pituitary adenomas were collected during surgery and established in culture as per our protocol (Pecori Giraldi et al 2011). Specimens were treated with 10 - 50 µM silibinin for up to 72 hours. ACTH medium levels were measured by Elisa; POMC expression was assessed by RT-PCR (Cassarino et al 2017). Results. Silibinin reduced spontaneous ACTH secretion to a variable extent in individual adenomas: from 32 to 79% of baseline at 4h, and 54 - 85 % of baseline at 48 and 72h. Silibinin was also effective in reinstating or enhancing sensitivity to steroid negative feedback: ACTH decreases during 10–50 µM silibinin incubation ranged from 10 to 63% of dexamethasone-treated wells at 4 hours, 70 -80% at 48 hours and 36 to 80% at 72 hours, indicating long-lasting effect on glucocorticoid sensitivity. Silibinin induced a variable decrease in POMC expression, both as regards expression in control and dexamethasone-treated wells; some specimens exhibited a marked sensitivity to the inhibitory effect, with POMC expression decreasing to less than 50% of control. Conclusions:, this data suggests that silibinin can inhibit ACTH secretion and POMC synthesis and restore sensitivity to negative glucocorticoid feedback.

SAT-282 Hypothalamic Pituitary Adrenal Axis Hyperactivity in Db/db Mice

Background: Patients with diabetes present hypercortisolemia, which may exacerbate not only glycemic control but also diabetic complications and fatty liver (1). Blood steroid hormone levels including corticosterone and aldosterone are high in db/db mice (2), although the mechanism is still unclear. Objective: To reveal the mechanism underlying the hypothalamic-pituitary-adrenal (HPA) axis hyperactivity in obese and diabetic condition. Method: Total RNA from hypothalamus, pituitary, and adrenal glands in ten-week-old db/db and db/+ mice were extracted and analyzed with DNA microarray and real-time PCR. Experiments were approved by the Institutional Animal Care and Use Committee. Results: Hypothalamic Crh mRNA expression was not changed in db/db mice, although Avp expression was 2.8-fold higher than db/+ mice. Pituitary Crhr1 and Pomc expression were higher (3.0- and 7.0-fold, respectively), and Nr3c1 expression was lower (0.7-fold) in db/db mice than db/+ mice. Adrenal Mc2r, Nr5a1, Cyp11b1, and Cyp11b2 were higher in db/db mice than db/+ mice (1.7-, 1.7-, 2.2-, 1.3-fold, respectively). Conclusion: Increased expression of tropic hormone receptors and abrogated negative feedback system may be responsible for HPA axis hyperactivity in db/db mice. Reference: (1) Chiodini I et al. Diabetes Care. 2007 Jan;30:83–8. (2) Hofmann A et al. Horm Metab Res. 2017 Jan;49:43–49.

SAT-299 Chronic Treatment Of Juvenile Hypothalamic Pomc-deficient Mice With RM-493 Prevents The Development Of Obesity

Arc-Pomc knockout mice have a disruption of the two neural enhancers for the Pomc (proopiomelanocortin) gene, resulting in selective loss of Pomc gene expression in the arcuate nucleus of the hypothalamus. This gene targeting strategy leaves pituitary Pomc expression unaffected. These mice are hyperphagic starting at weaning, and develop progressive obesity, infertility and insulin resistance over their lifetime. RM-493 (setmelanotide) is a melanocortin-4 receptor agonist that has shown promise in treating humans with Pomc null mutations. In this preclinical study, we investigated the effects of chronic RM-493 treatment using subcutaneously implanted osmotic minipumps in two groups of male mice: Arc-Pomc knockout mice, fed regular chow throughout the study period, and their wildtype counterparts, fed a 45% high-fat diet. Each of these groups of mice was randomized into three treatment cohorts at weaning: one that was given RM-493 throughout the entire study period (4–24 weeks of age, “RM-493” group), one that was given RM-493 only for the first 4 weeks of the study (4–8 weeks of age, “switch” group) and then switched to vehicle, and one cohort that received vehicle for the entire study (“vehicle” group). We serially measured body weight, food intake, body composition, glucose tolerance, insulin tolerance, and several measures of metabolism using the Comprehensive Lab Animal Monitoring System, including oxygen consumption, energy expenditure, ambulatory activity and lipid and glucose oxidation.Among other results, at the end of the study (24 weeks of age), Arc-Pomc knockout mice in the RM-493 group weighed significantly less than either the switch or vehicle groups (p<0.05). Arc-Pomc knockout mice on RM-493 also had higher energy expenditure when compared to the switch and vehicle groups (p<0.05). In addition, RM-493 improved the glucose-insulin index for Arc-Pomc knockout mice (p<0.05). According to our preliminary results, wildtype mice on high-fat diet, treated chronically with RM-493, did not differ in any of these measurements from their switch and vehicle groups.We conclude that the obesity syndrome caused by a loss of hypothalamic Pomc expression was completely blocked by RM-493 treatment started before the onset of obesity, with no apparent desensitization to the drug’s action over 20 weeks. However, the beneficial effects of a single month’s treatment were steadily reversed within one month after switching to vehicle treatment. In contrast to the dramatic effects of RM-493 in the genetic obesity syndrome, at this time, there does not appear to be any phenotypic changes in wild-type mice with RM-493 administration on the development of obesity or secondary metabolic disruptions in response to high-fat diet consumption.

SUN-207 Modelling Long Term Glucocorticoid-Induced HPA Axis Suppression in Mice

Glucocorticoids are prescribed for >3 months to 1% of the UK population. 10-50% of these long-term glucocorticoid treated patients develop persistent HPA axis suppression associated with mortality and morbidity. We have developed a mouse model of glucocorticoid-induced HPA axis dysfunction to determine the mechanisms resulting in persistent HPA axis suppression. Experiment 1: 36 C57BL/6 adult male mice received Dexamethasone (DEX,~10µg/day) or vehicle (CTL) via drinking water for 28 days, following which treatment was stopped and tissues were harvested at 0, 7 and 28 days. DEX suppressed waking serum corticosterone at days 0 and 7, recovering by day 28. Adrenal size remained lower 28 days following DEX withdrawal. DEX had no effect on whole pituitary pomc, nr3c1 or crhr1 expression, although avpr1b was increased at day 0. In the adrenal, hsd3b2 and cyp11a1 expression were reduced at time 0; normalising by 28 days. Experiment 2: 24 POMC-GFP male mice were treated as above. Tissues were collected at day 0 (n=6), 7 (n=3) and 10 (n=3) following withdrawal. Pooled corticotrophs (groups of 3) were isolated by FACS and RNA extracted for qPCR. DEX reduced corticotroph pomc expression at time 0 (x20 fold reduction), with x5 fold suppression at day 7, which recovered with evidence of compensation by day 10. DEX increased expression of avpr1b but not crhr1.CONCLUSION: 28 days dexamethasone treatment in mice suppresses the HPA axis. HPA suppression is evident 7 days following withdrawal of dexamethasone in the adrenal, corticotroph population and corticosterone production. Further analysis will determine mechanisms for delays in HPA axis recovery.

SAT-311 Role of SREBP in Regulation of Corticotroph Tumor ACTH Secretion and Cell Proliferation

Cushing Disease (CD) is a life-threatening condition with suboptimal medical treatment. To identify drugs that not only inhibit ACTH secretion to attain eucortisolemia but also inhibit tumor growth, we conducted a high throughput screen employing a novel “gain of signal” ACTH AlphaLISA assay. From a kinase inhibitor library containing 430 compounds, we identified the dual PI3K/HDAC inhibitor, CUDC-907, as a potent inhibitor of both in vitro and in vivo corticotroph tumor ACTH secretion and growth. By stepwise comparison of CUDC-907 with mono-functional PI3K and HDAC inhibitors, we demonstrated that CUDC-907 exerts its inhibitory effect on ACTH secretion primarily through its inhibition of HDAC activity at the POMC transcriptional level; while PI3K-mediated inhibition of corticotroph cell viability further contributes to reduced ACTH secretion. We also used RNA-seq to characterize the global transcriptiome changes associated with CUDC-907 treatment. Hierarchical clustering showed that 1432 differentially expressed genes (DEGs, p≤0.05 and fold-change≥1.5) were altered by CUDC-907 treatment in comparison to the vehicle-treated control cells. Gene ontology (GO) analysis of 456 downregulated and 976 upregulated DEGs revealed that the most enriched biological processes were cholesterol biosynthesis (GO:0006695, p=1.977e-17) and the type I interferon signaling pathway (GO: 0060337, p=4.928e-7) respectively. Further analysis demonstrated downregulation of the membrane-bound transcription factor sterol regulatory element binding proteins (SREBPs). Downregulation of SREBP-2 by CUDC-907 as well as the several other target enzymes in the cholesterol biosynthesis and uptake pathway including IDI2, NSDHL, MVD, and HMGCR, was confirmed by real-time PCR. To further characterize a role for SREBP-2 in regulation of corticotroph tumor ACTH secretion and proliferation, we employed siRNA targeting endogenous SREBP-2 (SREBP-2 mRNA, Control vs. siRNA 1±0.03 vs. 0.6±0.08, p<0.05), and demonstrated that knockdown of SREBP-2 not only inhibited POMC mRNA expression (POMC mRNA, 1±0.03 vs. 0.7±0.01, p<0.01), and ACTH secretion (ACTH (ng/mL) 29±0.4 vs. 23±0.3, p<0.005), but also suppressed cell proliferation (Relative Proliferation Rate, 1±0.01 vs. 0.7±0.01, p<0.005). This was further confirmed by overexpression of cleaved mature SREBP-2, which led to increased POMC expression and cell proliferation. We demonstrate for the first time the role of the SREBP-mediated cholesterol biosynthesis pathway in regulation of corticotroph tumor POMC regulation and growth. Our studies identify SREBP and cholesterol biosynthesis as a therapeutic target in CD.

MON-709 Single-Cell RNA-Sequencing Deciphers POMC Neuron Destiny

The hypothalamus is one of the critical brain nodes regulating body weight and energy homeostasis. Within this node, Pomc neurons sense nutrient and hormonal signals to release melanocortin peptides that induce satiety, whereas AgRP/NPY neurons exert opposite effects by releasing AGRP that promotes feeding. Immature neurons in the hypothalamic ventricular zone start to express Pomc at E10.5, reach a maximum number at E14.5 and then decrease to stabilize at E18.5. However, it remains elusive how Pomc expressing precursors adopt their final cell fates. Therefore, the goal of this study was to decipher the temporal sequence of transcription factor (TF) expression leading to the terminal differentiation of POMC neurons. Red fluorescent cells collected from dissociated hypothalami of Pomc-tDimer-dsRed mice at six critical developmental time points - E11.5, E13.5, E15.5, E17.5, P5 and P12- were FACS sorted for the 10X genomics scRNA-seq pipeline. Unsupervised cell clustering identified 11 distinct clusters based on their transcriptional profiles. Eight of the clusters were highly-enriched for neuronal signature genes and were further characterized based on their transcript levels for Pomc (high, medium or low) and other distinct feature genes. Cells in the Pomchigh cluster expressed genes identified previously to modulate Pomc expression, including Isl1, Nkx2-1, and Tbx3, together with several novel candidate TFs. Unexpectedly, Nr5a1, the ventromedial hypothalamic nucleus marker gene encoding SF1, was highly expressed in the Pomchigh cluster at early stages. One of the Pomclow clusters highly expressed Otp, Agrp, Npy, Sst and Calcr while a second was highly enriched with Tac2, Kiss1, Pdyn, Prlr, Ar and Esr1 transcripts. All the clusters showed direct correlations of embryonic stage with the expression of progressively more mature markers of differentiation, thereby extending previous reports of these clusters based on single time points. Moreover, our results uncovered five novel Pomc neuron clusters with unique patterns of TF gene expression. For comparison of these data to the adult hypothalamus, we performed a TRAP-Seq study using PomcCreERT,Rosa26eGFP-L10a mice. Prdm12 and Tbx3 were among the most highly differentially expressed TFs in the POMC neuron affinity purified translatome. Similarly, Cited1, Npy2r, and Asb4 were highly expressed in both the Pomchigh cluster and the TRAP-Seq derived POMC translatome. This comprehensive molecular characterization of POMC cells during development sheds new light on the molecular diversification of early POMC neuron precursors and provides a valuable resource for elucidating the regulatory mechanisms defining POMC neuron subgroups in the hypothalamus.

SAT-304 Pituitary Stem Cells May Drive Adenomas Causing Cushing’s Disease

Introduction:Cell rests of self-renewing Sox2+ progenitor cells have been identified in the normal pituitary glands1, however their role in human pituitary tumorigenesis is not understood. Adrenocorticotropic hormone (ACTH) producing microadenomas that cause Cushing’s disease frequently (~70%) lack pathogenic genetic mutations.2 In mice, targeted expression of oncogenic β-catenin in Sox2+ cells generate microadenomas. Interestingly, the Sox2+ cells reside within the adjacent normal gland and drive adenomas in a paracrine fashion.3 We hypothesized that Sox2+ progenitors in human pituitary gland may drive the formation of microadenomas that cause Cushing’s disease (CD). Methods:Four ACTH producing adenomas and two non-functional adenomas (NFPA) with separately annotated adjacent normal tissue (henceforward called ‘microenvironment’) were procured for this study (NCT00060541). We performed RNA deep sequencing (RNAseq) and compared expression of lineage-specific markers and progenitor markers using two-sample T-tests after testing for variance equality and using Welch’s approximation for degrees of freedom. Results:We found expected overexpression of ACTH preprohormone POMC in CD adenomas compared to adjacent microenvironment (?-fold) and NFPA (?-fold). The microenvironment in Cushing’s disease showed increased expression of progenitor markers including SOX2, SOX9, CDH1, GRFA2, and KLF4 compared with microenviroment in NFPA. Likewise, the Cushing’s disease microenvironment showed increased expression ofPOMC (26.98 - fold, P = 0.004) as well as PRLR (FC 17.39, P = 0.006) and GH1 (FC 29.91, P = 0.003) implying that increased Sox2+ progenitors contribute to terminally differentiated corticotrope, lactotroph and somatotroph lineages in-vivo. Conclusions:We report increased expression of several progenitor markers and concomitant elevation in tissues-specific markers in the microenvironment of Cushing’s disease patients. Our results indicate that increased pituitary progenitors in the microenvironment of human corticotropinomas may signal in paracrine fashion and may contribute to the pathogenesis of Cushing’s disease.

SAT-451 Chronic Stress During Adolescence of Rats Shows Sex Dimorphism in the Thyroid Axis Response to Voluntary Exercise in Adulthood

Exposure to chronic stress during adolescence causes long-term effects on the response of Hypothalamus-Pituitary-Adrenal (HPA) axis, affecting behavior and energy homeostasis. Voluntary exercise activates the HP-Thyroid (HPT) axis allowing efficient fluxes of substrates to active target organs. Chronic stress in adult rats blunts HPT axis response to voluntary exercise in a sex-dependent manner (Front Endocrinol 10(418):1-13, 2019). As adolescents show sex-dependent responses to stress, we sought to evaluate the effect of chronic stress at this period in the response of HPT axis to voluntary exercise in adulthood. Wistar male and female rats were divided in an undisturbed group (Control, C) and one group exposed to chronic variable stress (CVS) where the rats were daily subjected to different stressors during postnatal day (PND) 30 to 60 for females and PND 30 to 70 for males. At adulthood (PND 74 for females and PND 84 for males) rats were exposed to running wheel following published protocol (Endocrinology 155:2020-2030, 2014). As females are more susceptible to stress during adolescence than males, additional independent experiments were performed with female rats kept in group or individual housing, since PND 30 (2 per cage or isolated (Iso)). At PND 64, Iso rats were housed in pairs and exposed to CVS every 3 days until PND 80; later, rats were exercised 26 days. Hormones were quantified by ELISA or RIA; mRNA expression was determined by RT-PCR. Voluntary exercise reduced fat mass in C groups, dependent on the amount of exercise performed; stressed rats exercised did not lose fat, indicating that adolescent stress avoids an appropriate energy distribution during exercise. The expression of Crh and Avp in hypothalamic paraventricular nucleus (PVN) decreased in stressed groups mainly in females, as reported. Exercise decreased corticosterone levels only in C rats, suggesting that CVS during adolescence modifies the HPA axis response to exercise. CVS inhibited Pomc expression induced by exercise and increased Npy expression in arcuate nucleus, decreased Trh expression in PVN for both sexes and in dorsomedial hypothalamus in males. Thyroid hormones were not altered in CVS males and Iso females; however, T3 and T4 levels were high in CVS females, so different stress exposures may modify the HPT axis state in females. The response to exercise of the target organs of thyroid hormones reveals with more accuracy the activity of HPT axis, exercise stimulated the expression of Adrb3 and Dio2 in brown adipose tissue of C females, and the expression of Dio2 and Pgc1a in skeletal muscle (gastrocnemius) of both sexes, changes attenuated by CVS. These results indicate that chronic stress during adolescence blunts the response of HPT axis to voluntary exercise, strongly in females than males.

Maternal exercise during gestation and lactation decreases high-fat diet preference by altering central reward system gene expression in adult female offspring from high-fat fed dams

Exposure to maternal high-fat (HF) diet during gestation and lactation alters adult offspring’s feeding behavior and diet preference. However, the impact of maternal exercise on offspring’s diet preference and reward system development is less studied. In this study, we investigate the effect of perinatal maternal exercise on the development of diet preference, dopamine- and opioid-related gene expression in the central reward system in female offspring from HF-fed Sprague-Dawley rat dams. We found maternal HF diet did not alter adult offspring HF preference, but influenced offspring’s dopamine and opioid system both at weaning and in adulthood, and these offspring retained higher body weight in adulthood. However, offspring from dams exposed to both HF diet and exercise during gestation and lactation had normalized body weight, decreased fat mass and lower HF-diet preference but increased energy intake in adulthood. The dopamine- and opioid-related gene expression in central reward system and POMC expression in hypothalamus was elevated in these adult offspring. We conclude that maternal exercise during gestation and lactation can potentially overcome the negative effects of perinatal exposure to HF diet in female offspring by altering their diet preference, central reward system signaling and hypothalamus neuropeptide expression.

Hypothalamic Pomc expression restricted to GABAergic neurons suppresses Npy overexpression and restores food intake in obese mice

ObjectiveHypothalamic arcuate proopiomelanocortin (Arc-POMC) neurons are involved in different physiological processes such as the regulation of energy balance, glucose homeostasis, and stress-induced analgesia. Since these neurons heterogeneously express different biological markers and project to many hypothalamic and extrahypothalamic areas, it is proposed that Arc-POMC neurons could be classified into different subpopulations having diverse physiological roles. The aim of the present study was to characterize the contribution of the subpopulation of Arc-POMC neurons cosecreting gamma-aminobutyric acid (GABA) neurotransmitter in the control of energy balance. MethodsArc-Pomc expression restricted to GABAergic-POMC neurons was achieved by crossing a reversible Pomc-deficient mouse line (arcPomc−) with a tamoxifen-inducible Gad2-CreER transgenic line. Pomc expression was rescued in the compound arcPomc−/−:Gad2-CreER female and male mice by tamoxifen treatment at postnatal days 25 (P25) or 60 (P60), and body weight, daily food intake, fasting glycemia, and fasting-induced hyperphagia were measured. POMC recovery was quantified by immunohistochemistry and semiquantitative RT-PCR. Neuropeptide Y (NPY) and GABAergic neurons were identified by in situ hybridization. Arc-POMC neurons projecting to the dorsomedial hypothalamic nucleus (DMH) were studied by stereotactic intracerebral injection of fluorescent retrobeads into the DMH. ResultsTamoxifen treatment of arcPomc−/−:Gad2-CreER mice at P60 resulted in Pomc expression in ∼23–25% of Arc-POMC neurons and ∼15–23% of Pomc mRNA levels, compared to Gad2-CreER control mice. Pomc rescue in GABAergic-POMC neurons at P60 normalized food intake, glycemia, and fasting-induced hyperphagia, while significantly reducing body weight. Energy balance was also improved in arcPomc−/−:Gad2-CreER mice treated with tamoxifen at P25. Distribution analysis of rescued POMC immunoreactive fibers revealed that the DMH is a major target site of GABAergic-POMC neurons. Further, the expression of the orexigenic neuropeptide Y (NPY) in the DMH was increased in arcPomc−/− obese mice but was completely restored after Pomc rescue in arcPomc−/−:Gad2-CreER mice. Finally, we found that ∼75% of Arc-POMC neurons projecting to the DMH are GABAergic. ConclusionsIn the present study, we show that the expression of Pomc in the subpopulation of Arc-GABAergic-POMC neurons is sufficient to maintain normal food intake. In addition, we found that DMH-NPY expression is negatively correlated with Pomc expression in GABAergic-POMC neurons, suggesting that food intake may be regulated by an Arc-GABAergic-POMC → DMH-NPY pathway.

Expression of a hypomorphic Pomc allele alters leptin dynamics during late pregnancy.

Proopiomelanocortin (POMC) neurons in the hypothalamic arcuate nucleus (ARC) are essential for normal energy homeostasis. Maximal ARC Pomc transcription is dependent on neuronal Pomc enhancer 1 (nPE1), located 12 kb upstream from the promoter. Selective deletion of nPE1 in mice decreases ARC Pomc expression by 70%, sufficient to induce mild obesity. Because nPE1 is located exclusively in the genomes of placental mammals, we questioned whether its hypomorphic mutation would also alter placental Pomc expression and the metabolic adaptations associated with pregnancy and lactation. We assessed placental development, pup growth, circulating leptin and expression of Pomc, Agrp and alternatively spliced leptin receptor (LepR) isoforms in the ARC and placenta of Pomc∆1/∆1 and Pomc+/+ dams. Despite indistinguishable body weights, lean mass, food intake, placental histology and Pomc expression and overall pregnancy outcomes between the genotypes, Pomc ∆1/∆1 females had increased pre-pregnancy fat mass that paradoxically decreased to control levels by parturition. However, Pomc∆1/∆1 dams had exaggerated increases in circulating leptin, up to twice of that of the typically elevated levels in Pomc+/+ mice at the end of pregnancy, despite their equivalent fat mass. Pomc∆1/∆1dams also had increased placental expression of soluble leptin receptor (LepRe), although the protein levels of LEPRE in circulation were the same as Pomc+/+ controls. Together, these data suggest that the hypomorphic Pomc∆1/∆1 allele is responsible for the perinatal super hyperleptinemia of Pomc∆1/∆1 dams, possibly due to upregulated leptin secretion from individual adipocytes.

Effects of gut microbiota on leptin expression and body weight are lessened by high-fat diet in mice.

Aberration in leptin expression is one of the most frequent features in the onset and progression of obesity, but the underlying mechanisms are still unclear and need to be clarified. This study investigated the effects of the absence of gut microbiota on body weight and the expression and promoter methylation of the leptin. Male C57 BL/6 J germ-free (GF) and conventional (CV) mice (aged 4-5 weeks) were fed either a normal-fat diet (NFD) or a high-fat diet (HFD) for 16 weeks. Six to eight mice from each group, at 15 weeks, were administered exogenous leptin for 7 d. Leptin expression and body weight gain in GF mice were increased by NFD with more CpG sites hypermethylated at the leptin promoter, whereas there was no change with HFD, compared with CV mice. Adipose or hepatic expression of genes associated with fat synthesis (Acc1, Fas and Srebp-1c), hydrolysis and oxidation (Atgl, Cpt1a, Cpt1c, Ppar-α and Pgc-1α) was lower, and hypothalamus expression of Pomc and Socs3 was higher in GF mice than levels in CV mice, particularly with NFD feeding. Exogenous leptin reduced body weight in both types of mice, with a greater effect on CV mice with NFD. Adipose Lep-R expression was up-regulated, and hepatic Fas and hypothalamic Socs3 were down-regulated in both types of mice. Expression of fat hydrolysis and oxidative genes (Atgl, Hsl, Cpt1a, Cpt1c, Ppar-α and Pgc-1α) was up-regulated in CV mice. Therefore, the effects of gut microbiota on the leptin expression and body weight were affected by dietary fat intake.

Seasonal reproductive state determines gene expression in the hypothalamus of a latitudinal migratory songbird during the spring and autumn migration

We investigated gonadal effects on hypothalamic transcription of genes in sham-operated and castrated redheaded buntings photostimulated into spring and autumn migratory states. RNA-Seq results showed testes-dependent differences between spring and autumn migratory states. In particular, differentially expressed genes enriched G-protein-coupled receptor and calcium-ion signaling pathways during spring and autumn states, respectively. qPCR assay showed attenuated gabra5, ttr, thra and thrb expressions, suggesting reduced GABA and thyroid hormone effects on photo-sexual response in spring. In spring castrates, reduced npy, tac1 and nrcam and increased ank3 expression suggested testicular effects on the appetite, prolactin release and neuronal functions, whereas in autumn castrates, reduced rasgrp1, grm5 and grin1, and increased mras expression suggested testicular effects on the ras, G-protein and glutamate signaling pathways. Castration-induced reciprocal switching of pomc and pdyn expressions suggested effects on the overall homeostasis in both seasons. These results demonstrate transcriptome-wide changes, with season-dependent roles of testes in songbird migration.

POMC gene expression, polymorphism, and the association with reproduction traits in chickens

Reproduction trait is one of the most important economic traits in poultry industry. This study was aimed to investigate the mRNA expression levels, single nucleotide polymorphisms (SNP) of POMC gene, and the association with reproduction traits in chickens. Five SNP (g.958 G > A, g.1374 G > C, g.1393 G > A, g.1817 C > T, and g.1918G > A) were detected in introns of POMC gene in 317 Zhenning yellow chickens. Association analysis revealed that g.958 G > A and g.1817 C > T showed significantly associations with fertilization rate, hatching rate of hatching eggs, and hatching rate of fertilized eggs in chickens. Simultaneously, g.1374 G > C and g.1918G > A were both associated with egg weight at 300 D of age (P < 0.05). The SNP of g.958 G > A, g.1393 G > A, and g.1817 C > T were all associated with E2 hormone levels (P < 0.05). The result of mRNA expression levels in different tissues showed that POMC mRNA expression level in the pituitary was higher than those in the other tissues and varied in different genotypes. In conclusion, the results in this study provided new evidences that polymorphisms of the POMC gene have potential effects on reproduction traits in chickens. The 5 SNP detected in this study could be potential markers for improving reproduction traits in chickens.

Role of leptin in the regulation of food intake in fasted mice.

Leptin is well acknowledged as an anorexigenic hormone that plays an important role in feeding control. Hypothalamic GABA system plays a significant role in leptin regulation on feeding and metabolism control. However, the pharmacological relationship of leptin and GABA receptor is still obscure. Therefore, we investigated the effect of leptin or combined with baclofen on the food intake in fasted mice. We detected the changes in hypothalamic c‐Fos expression, hypothalamic TH, POMC and GAD67 expression, plasma insulin, POMC and GABA levels to demonstrate the mechanisms. We found that leptin inhibit fasting‐induced increased food intake and activated hypothalamic neurons. The inhibitory effect on food intake induced by leptin in fasted mice can be reversed by pretreatment with baclofen. Baclofen reversed leptin's inhibition on c‐Fos expression of PAMM in fasted mice. Therefore, these results indicate that leptin might inhibit fasting‐triggered activation of PVN neurons via presynaptic GABA synaptic functions which might be partially blocked by pharmacological activating GABA‐B. Our findings identify the role of leptin in the regulation of food intake.

Early weaning modulates eating behavior and promotes hypofunction of the serotonergic (5HT) system in juvenile male rats.

Early life stress (ELS) has been associated with developmental impairments. Early weaning (EW)is a postnatal stress model consisting of interruption of lactation and maternal care. The 5HT-system has been associated with neurobehavioral modulations promoted by ELS. Thus, the present work aims to investigate the effects of early weaning on feeding behavior and serotonergic system of juvenile male rats. For this, rats were submitted to early (PND15) or natural (PND30) weaning and had the body weight, food intake in circadian phases, and food intake in response to fenfluramine assessed. mRNA expression of serotoninergic receptors (5HT1A and 5HT2C) and transporter (SERT) was assessed in the hypothalamus and brainstem, as well as NPY and POMC mRNA expression in hypothalamus. The results show that early weaning promoted changes in the percentage of weight gain during lactation period and increase in body weight at PND40. It was also observed that EW promoted increase and decrease in food intake in light and dark phase, respectively, and leads to a decreased action of fenfluramine on inhibition of food intake. In addition, early weaning promoted increased NPY and SERT mRNA expression in the hypothalamus and 5HT2C in the brainstem. Together, the data indicate that the stress caused by early weaning impairs the eating behavior of juvenile male rats through hypofunction of the 5HT-system.