Alx3 deficiency disrupts energy homeostasis, alters body composition, and impairs hypothalamic regulation of food intake.

The coordination of food intake, energy storage, and expenditure involves complex interactions between hypothalamic neurons and peripheral tissues including pancreatic islets, adipocytes, muscle, and liver. Previous research shows that deficiency of the transcription factor Alx3 alters pancreatic islet-dependent glucose homeostasis. In this study we carried out a comprehensive assessment of metabolic alterations in Alx3 deficiency. We report that Alx3-deficient mice exhibit decreased food intake without changes in body weight, along with reduced energy expenditure and altered respiratory exchange ratio. Magnetic resonance imaging reveals increased adiposity and decreased muscle mass, which was associated with markers of motor and sympathetic denervation. By contrast, Alx3-deficient mice on a high-fat diet show attenuated weight gain and improved insulin sensitivity, compared to control mice. Gene expression analysis demonstrates altered lipogenic and lipolytic gene profiles. In wild type mice Alx3 is expressed in hypothalamic arcuate nucleus neurons, but not in major peripheral metabolic organs. Functional diffusion-weighted magnetic resonance imaging reveals selective hypothalamic responses to fasting in the arcuate nucleus of Alx3-deficient mice. Additionally, altered expression of proopiomelanocortin and melanocortin-3 receptor mRNA in the hypothalamus suggests impaired regulation of feeding behavior. This study highlights the crucial role for Alx3 in governing food intake, energy homeostasis, and metabolic nutrient partitioning, thereby influencing body mass composition.

The homeodomain transcription factor Six3 regulates hypothalamic Pomc expression and its absence from POMC neurons induces hyperphagia and mild obesity in male mice.

OBJECTIVE: Proopiomelanocortin (POMC) neurons release potent anorexigenic neuropeptides, which suppress food intake and enhance energy expenditure via melanocortin receptors. Although the importance of central melanocortin in physiological regulation is well established, the underlying genetic mechanisms that define the functional identity of melanocortin neurons and maintain hypothalamic Pomc expression remain to be fully determined. In this study, we investigate the functional significance of Six3, a transcriptional regulator notably expressed in embryonic and adult mouse POMC neurons, in the regulation of hypothalamic Pomc expression and downstream physiological consequences. METHODS: We first evaluated the expression of Six3 in the developing and adult hypothalamus by double fluorescence in situ hybridization. Next, we assessed POMC immunoreactivity in mutant mice selectively lacking Six3 from Pomc-expressing neurons and quantified Pomc mRNA levels in a tamoxifen-inducible Six3 knockout mouse model activated at embryonic E9.5 days. We also determined glucose and insulin sensitivity, daily food intake, body composition and body weight in adult male and female mice lacking Six3 specifically from POMC neurons. Lastly, we assessed the physiological consequences of ablating Six3 from POMC neurons in adult mice. RESULTS: Six3 and Pomc were co-expressed in mouse hypothalamic neurons during development and adulthood. Mouse embryos deficient in Six3 showed reduced Pomc expression in the developing hypothalamus. Targeted deletion of Six3 specifically from POMC neurons resulted in decreased hypothalamic Pomc expression, increased daily food intake, enhanced glucose sensitivity and mild obesity in male but not in female mice. Finally, conditional removal of Six3 from POMC neurons in adult mice led to a reduction in hypothalamic POMC immunoreactivity with no significant effects in body weight or food intake. CONCLUSIONS: Altogether, our results demonstrate that Six3 plays an essential role in the early establishment of POMC neuron identity and the maintenance of physiological levels of hypothalamic Pomc expression. In addition, our study demonstrates that the functional significance of Six3 expression in POMC neurons is sexually dimorphic and age-dependent.

Hypothalamic GABAergic Neurons Expressing Cellular Retinoic Acid Binding Protein 1 (CRABP1) Are Sensitive to Metabolic Status and Liraglutide in Male Mice.

INTRODUCTION: Owing to their privileged anatomical location, neurons of the arcuate nucleus of the hypothalamus (ARC) play critical roles in sensing and responding to metabolic signals such as leptin and glucagon-like peptide 1 (GLP-1). In addition to the well-known proopiomelanocortin (POMC)- and agouti-related peptide (AgRP)-expressing neurons, subpopulations of GABAergic neurons are emerging as key regulators of energy balance. However, the precise identity of these metabolic neurons is still elusive. Here, we identified and characterized the molecular signature of a novel population of GABAergic neurons of the ARC expressing Cellular retinoic acid binding protein 1 (Crabp1). METHODS: Using a combination of immunohistochemistry and in situ hybridization techniques, we investigated the expression of Crabp1 across the mouse brain and characterized the molecular identity of Crabp1ARC neurons. We also determined whether Crabp1ARC neurons are sensitive to fasting, leptin, and GLP1R agonism by assessing cFOS immunoreactivity as a marker of neuronal activity. RESULTS: Crabp1ARC neurons represent a novel GABAergic neuronal population robustly enriched in the ARC and are distinct from the prototypical melanocortin neurons. Crabp1ARC neurons overlap with three subpopulations of yet uncharacterized ARC neurons expressing Htr3b, Tbx19, and Tmem215. Notably, Crabp1ARC neurons express receptors for metabolic hormones and their activity is modulated by the nutritional state and GLP1R agonism. CONCLUSION: Crabp1ARC neurons represent a novel heterogeneous population of GABAergic neurons sensitive to metabolic status.

Transcriptomic analysis reveals the effects of maternal exposure to bisphenol AF on hypothalamic development in male neonatal mice.

Fragmented data suggest that bisphenol AF (BPAF), a chemical widely used in a variety of products, might have potential impacts on the hypothalamus. Here, we employed male neonatal mice following maternal exposure to explore the effects of low-dose BPAF on hypothalamic development by RNA-sequencing. We found that maternal exposure to approximately 50 µg/(kg·day) BPAF from postanal day (PND) 0 to PND 15 altered the hypothalamic transcriptome, primarily involving the pathways and genes associated with extracellular matrix (ECM) and intercellular adhesion, neuroendocrine regulation, and neurological processes. Further RNA analysis confirmed the changes in the expression levels of concerned genes. Importantly, we further revealed that low-dose BPAF posed a stimulatory impact on pro-opiomelanocortin (POMC) neurons in the arcuate nucleus of the hypothalamus and induced the browning of inguinal white adipose tissue. All findings indicate that developmental exposure to low-dose BPAF could interfere with hypothalamic development and thereby lead to alterations in the metabolism. Interestingly, 5000 µg/(kg·day) BPAF caused slighter, non-significant or even inverse alterations than the low dose of 50 µg/(kg·day), displaying a dose-independent effect. Further observations suggest that the the dose-independent effects of BPAF might be associated with oxidative stress and inflammatory responses caused by the high dose. Overall, our study highlights a risk of low-dose BPAF to human neuroendocrine regulation and metabolism.

The Role of Pro-Opiomelanocortin Derivatives in the Development of Type 2 Diabetes-Associated Myocardial Infarction: Possible Links with Prediabetes.

Myocardial infarction is a major contributor to CVD-related mortality. T2DM is a risk factor for MI. Stress activates the HPA axis, SNS, and endogenous OPS. These POMC derivatives increase the blood glucose and cardiovascular response by inhibiting the PI3K/AkT insulin signaling pathway and increasing cardiac contraction. Opioids regulate the effect of the HPA axis and SNS and they are cardioprotective. The chronic activation of the stress response may lead to insulin resistance, cardiac dysfunction, and MI. Stress and T2DM, therefore, increase the risk of MI. T2DM is preceded by prediabetes. Studies have shown that prediabetes is associated with an increased risk of MI because of inflammation, hyperlipidemia, endothelial dysfunction, and hypertension. The HPA axis is reported to be dysregulated in prediabetes. However, the SNS and the OPS have not been explored during prediabetes. The effect of prediabetes on POMC derivatives has yet to be fully explored and understood. The impact of stress and prediabetes on the cardiovascular response needs to be investigated. This study sought to review the potential impact of prediabetes on the POMC derivatives and pathways that could lead to MI.

POMC Neuron BBSome Regulation of Body Weight is Independent of its Ciliary Function.

The BBSome, a complex of several Bardet-Biedl syndrome (BBS) proteins including BBS1, has emerged as a critical regulator of energy homeostasis. Although the BBSome is best known for its involvement in cilia trafficking, through a process that involve BBS3, it also regulates the localization of cell membrane receptors underlying metabolic regulation. Here, we show that inducible Bbs1 gene deletion selectively in proopiomelanocortin (POMC) neurons cause a gradual increase in body weight, which was associated with higher fat mass. In contrast, inducible deletion of Bbs3 gene in POMC neurons failed to affect body weight and adiposity. Interestingly, loss of BBS1 in POMC neurons led to glucose intolerance and insulin insensitivity, whereas BBS3 deficiency in these neurons is associated with slight impairment in glucose handling, but normal insulin sensitivity. BBS1 deficiency altered the plasma membrane localization of serotonin 5-HT2C receptor (5-HT2CR) and ciliary trafficking of neuropeptide Y2 receptor (NPY2R).In contrast, BBS3 deficiency, which disrupted the ciliary localization of the BBSome, did not interfere with plasma membrane expression of 5-HT2CR, but reduced the trafficking of NPY2R to cilia. We also show that deficiency in BBS1, but not BBS3, alters mitochondria dynamics and decreased total and phosphorylated levels of dynamin-like protein 1 (DRP1) protein. Importantly, rescuing DRP1 activity restored mitochondria dynamics and localization of 5-HT2CR and NPY2R in BBS1-deficient cells. The contrasting effects on energy and glucose homeostasis evoked by POMC neuron deletion of BBS1 versus BBS3 indicate that BBSome regulation of metabolism is not related to its ciliary function in these neurons.

Review of the role of the endogenous opioid and melanocortin systems in the restless legs syndrome.

Restless legs syndrome (RLS) is responsive to opioid, dopaminergic and iron-based treatments. Receptor blocker studies in RLS patients suggest that the therapeutic efficacy of opioids is specific to the opioid receptor and mediated indirectly through the dopaminergic system. An RLS autopsy study reveals decreases in endogenous opioids, ß-endorphin and perhaps Met-enkephalin in the thalamus of RLS patients. A total opioid receptor knock-out (mu, delta and kappa) and a mu-opioid receptor knock-out mouse model of RLS show circadian motor changes akin to RLS and, although both models show sensory changes, the mu-opioid receptor knock mouse shows circadian sensory changes closest to those seen in idiopathic RLS. Both models show changes in striatal dopamine, anaemia and low serum iron. However, only in the total receptor knock-out mouse do we see the decreases in serum ferritin that are normally found in RLS. There are also decreases in serum iron when wild-type mice are administered a mu-opioid receptor blocker. In addition, the mu-opioid receptor knock-out mouse also shows increases in striatal zinc paralleling similar changes in RLS. Adrenocorticotropic hormone and α-melanocyte stimulating hormone are derived from pro-opiomelanocortin as is ß-endorphin. However, they cause RLS-like symptoms and periodic limb movements when injected intraventricularly into rats. These results collectively suggest that an endogenous opioid deficiency is pathogenetic to RLS and that an altered melanocortin system may be causal to RLS as well.

Dynamic, sex- and diet-specific pleiotropism in the PAC1 receptor-mediated regulation of arcuate proopiomelanocortin and Neuropeptide Y/Agouti related peptide neuronal excitability by anorexigenic ventromedial nucleus PACAP neurons.

This study furthers the investigation of how pituitary adenylate cyclase activating polypeptide (PACAP) and the PAC1 receptor (PAC1R) regulate the homeostatic energy balance circuitry. We hypothesized that apoptotic ablation of PACAP neurones in the hypothalamic ventromedial nucleus (VMN) would affect both energy intake and energy expenditure. We also hypothesized that selective PAC1R knockdown would impair the PACAP-induced excitation in anorexigenic proopiomelanocortin (POMC) neurones and inhibition of orexigenic neuropeptide Y (NPY)/agouti-related peptide (AgRP) neurones in the hypothalamic arcuate nucleus (ARC). The results show CASPASE-3-induced ablation of VMN PACAP neurones leads to increased energy intake and meal frequency as well as decreased energy expenditure in lean animals. The effects were more robust in obese males, whereas we saw the opposite effects in obese females. We then utilized visualized whole-cell patch clamp recordings in hypothalamic slices. PAC1R knockdown in POMC neurones diminishes the PACAP-induced depolarization, increase in firing, decreases in energy intake and meal size, as well as increases in CO2 production and O2 consumption. Similarly, the lack of expression of the PAC1R in NPY/AgRP neurones greatly attenuates the PACAP-induced hyperpolarization, suppression of firing, decreases in energy intake and meal frequency, as well as increases in energy expenditure. The PACAP response in NPY/AgRP neurones switched from predominantly inhibitory to excitatory in fasted animals. Finally, the anorexigenic effect of PACAP was potentiated when oestradiol was injected into the ARC in ovariectomized females. This study demonstrates the critical role of anorexigenic VMN PACAP neurones and the PAC1R in exciting POMC and inhibiting NPY/AgRP neurons to control homeostatic feeding.

Cotadutide (GLP-1/Glucagon dual receptor agonist) modulates hypothalamic orexigenic and anorexigenic neuropeptides in obese mice.

The hypothalamic neuropeptides linked to appetite and satiety were investigated in obese mice treated with cotadutide (a dual receptor agonist of glucagon-like peptide 1 (GLP-1R)/Glucagon (GCGR)). Twelve-week-old male C57BL/6 mice were fed a control diet (C group, n = 20) or a high-fat diet (HF group, n = 20) for ten weeks. Each group was further divided, adding cotadutide treatment and forming groups C, CC, HF, and HFC for four additional weeks. The hypothalamic arcuate neurons were labeled by immunofluorescence, and protein expressions (Western blotting) for neuropeptide Y (NPY), proopiomelanocortin (POMC), agouti-related protein (AgRP), and cocaine- and amphetamine-regulated transcript (CART). Cotadutide enhanced POMC and CART neuropeptides and depressed NPY and AGRP neuropeptides. In addition, gene expressions (RT-qPCR) determined that Lepr (leptin receptor) and Calcr (calcitonin receptor) were diminished in HF compared to C but enhanced in CC compared to C and HFC compared to HF. Besides, Socs3 (suppressor of cytokine signaling 3) was decreased in HFC compared to HF, while Sst (somatostatin) was higher in HFC compared to HF; Tac1 (tachykinin 1) and Mc4r (melanocortin-4-receptor) were lower in HF compared to C but increased in HFC compared to HF. Also, Glp1r and Gcgr were higher in HFC compared to HF. In conclusion, the findings are compelling, demonstrating the effects of cotadutide on hypothalamic neuropeptides and hormone receptors of obese mice. Cotadutide modulates energy balance through the gut-brain axis and its associated signaling pathways. The study provides insights into the mechanisms underlying cotadutide's anti-obesity effects and its possible implications for obesity treatment.

HFD-exacerbated Metabolic Side Effects of Olanzapine Are Suppressed by ER Stress Inhibitor.

OBJECTIVE: Numerous schizophrenic patients are suffering from obesity primarily attributed to antipsychotic medication and poor dietary habits. This study investigated the progressive deterioration of olanzapine-induced metabolic disorders in the presence of a high-fat diet (HFD) and explored the involvement of endoplasmic reticulum (ER) stress. METHODS: Female Sprague-Dawley rats fed on a standard chow diet or HFD were treated with olanzapine (3 mg/kg/day) and the ER stress inhibitor 4-phenylbutyric acid (4-PBA, 1 and 0.5 g/kg/day) for 8 days. Changes in body weight, food intake, and plasma lipids were assessed. Hepatic fat accumulation was evaluated using oil red O staining. Western blotting and immunofluorescence assays were employed to examine the expression of ER stress markers, NOD-like receptor pyrin domain-containing protein 3 (NLRP3), and proopiomelanocortin (POMC) in the hypothalamus or liver. RESULTS: Compared to olanzapine alone, olanzapine+HFD induced greater weight gain, increased hyperlipidemia, and enhanced hepatic fat accumulation (P<0.05). Co-treatment with 4-PBA exhibited a dose-dependent inhibition of these effects (P<0.05). Further mechanistic investigations revealed that olanzapine alone activated ER stress, upregulated NLRP3 expression in the hypothalamus and liver, and downregulated hypothalamic POMC expression. The HFD exacerbated these effects by 50%-100%. Moreover, co-administration of 4-PBA dose-dependently attenuated the olanzapine+HFD-induced alterations in ER stress, NLRP3, and POMC expression in the hypothalamus and liver (P<0.05). CONCLUSION: HFD worsened olanzapine-induced weight gain and lipid metabolic disorders, possibly through ER stress-POMC and ER stress-NLRP3 signaling. ER stress inhibitors could be effective in preventing olanzapine+HFD-induced metabolic disorders.

Cushing Syndrome due to a Pancreatic Neuroendocrine Tumor Treated With Radiofrequency Ablation.

Delayed diagnosis of Cushing syndrome (CS) results in advanced disease, treatment delays, and poor outcomes. We present a patient with ectopic ACTH syndrome (EAS) from a pancreatic neuroendocrine tumor (NET) whose care posed diagnostic and therapeutic challenges. A 59-year-old female with classic Cushing stigmata, biochemical evidence of ACTH-dependent hypercortisolism, and a 5-mm pituitary lesion presented for inferior petrosal sinus sampling, which was contraindicated due to non-ST elevation myocardial infarction and acute/subacute strokes. Whole-body computed tomography (CT) scan was unrevealing, but elevations in chromogranin A and proopiomelanocortin (POMC) concentrations suggested EAS. Positron emission tomography-CT with gallium 68-DOTATATE demonstrated a 7-mm pancreatic tail lesion, suspicious for a pancreatic NET. The patient was not a surgical candidate and treatment with ketoconazole was complicated by hepatoxicity. Endoscopic ultrasound-guided biopsy and radiofrequency ablation of the lesion was pursued. Pathology confirmed ACTH immunoreactive low-grade pancreatic NET. Post procedure, sustained normalization of ACTH and cortisol was achieved. This case supports the utility of POMC measurements in the differential diagnosis of CS and the use of advanced nuclear imaging for tumor localization. For patients with functional pancreatic NET who are poor surgical candidates or intolerant of pharmacotherapy, novel endoscopic ablation may offer a low-risk therapeutic option and should be further investigated.

Beneficial Effects of Setmelanotide in a 5-Year-Old Boy With POMC Deficiency and on His Caregivers.

The melanocortin-4 receptor agonist setmelanotide is now recommended for the treatment of genetic obesity due to proopiomelanocortin (POMC), proprotein convertase subtilisin/kexin type 1 (PCSK1), or leptin receptor (LEPR) deficiency in patients aged 6 years and older. Here, we describe the clinical benefit of setmelanotide administration in a 5-year-old child with severe hyperphagia and obesity due to POMC deficiency. Daily administration of 0.5 mg setmelanotide for 12 months resulted in significant weight loss of -30 kg from baseline (-36% of weight loss) and improvements in hyperphagia and metabolic status. No major side effects were observed, except for hyperpigmentation and transient spontaneous erections. Interestingly, the clinical improvement of the child was associated with a remarkable improvement in the quality of life of the parents, along with a decrease in their emotional scores. This observation supports the early use of setmelanotide in young children with melanocortin pathway variants, in order to limit the adverse consequences of early and extreme weight gain, and to improve the quality of life of patients and of their relatives.

Management of Monogenic and Syndromic Obesity.

Similar to the general population, lifestyle interventions focused on nutrition and physical activity form the foundation for treating obesity caused by rare genetic disorders. Additional therapies, including metreleptin and setmelanotide, that target defects within the leptin signaling pathway can effectively synergize with lifestyle efforts to treat monogenic disorders of leptin, leptin receptor, proopiomelanocortin (POMC), and proprotein convertase subtilisin/kexin type 1 (PCSK1) and syndromic conditions, such as the ciliopathies Bardet-Biedl and Alström syndromes, whose pathophysiological mechanisms also converge on the leptin pathway. Investigational treatments for Prader-Willi syndrome target specific defects caused by reduced expression of paternally derived genes within the chromosome 15q region.

Marchf6 E3 ubiquitin ligase critically regulates endoplasmic reticulum stress, ferroptosis, and metabolic homeostasis in POMC neurons.

The metabolic prohormone pro-opiomelanocortin (POMC) is generally translocated into the endoplasmic reticulum (ER) for entry into the secretory pathway. Patients with mutations within the signal peptide (SP) of POMC or its adjoining segment develop metabolic disorders. However, the existence, metabolic fate, and functional outcomes of cytosol-retained POMC remain unclear. Here, we show that SP-uncleaved POMC is produced in the cytosol of POMC neuronal cells, thus inducing ER stress and ferroptotic cell death. Mechanistically, the cytosol-retained POMC sequesters the chaperone Hspa5 and subsequently accelerates degradation of the glutathione peroxidase Gpx4, a core regulator of ferroptosis, via the chaperone-mediated autophagy. We also show that the Marchf6 E3 ubiquitin ligase mediates the degradation of cytosol-retained POMC, thereby preventing ER stress and ferroptosis. Furthermore, POMC-Cre-mediated Marchf6-deficient mice exhibit hyperphagia, reduced energy expenditure, and weight gain. These findings suggest that Marchf6 is a critical regulator of ER stress, ferroptosis, and metabolic homeostasis in POMC neurons.

Agouti-Signalling Protein Overexpression Reduces Aggressiveness in Zebrafish.

Feeding motivation plays a crucial role in food intake and growth. It closely depends on hunger and satiation, which are controlled by the melanocortin system. Overexpression of the inverse agonist agouti-signalling protein (ASIP) and agouti-related protein (AGRP) leads to enhanced food intake, linear growth, and weight. In zebrafish, overexpression of Agrp leads to the development of obesity, in contrast to the phenotype observed in transgenic zebrafish that overexpress asip1 under the control of a constitutive promoter (asip1-Tg). Previous studies have demonstrated that asip1-Tg zebrafish exhibit larger sizes but do not become obese. These fish display increased feeding motivation, resulting in a higher feeding rate, yet a higher food ration is not essential in order to grow larger than wild-type (WT) fish. This is most likely attributed to their improved intestinal permeability to amino acids and enhanced locomotor activity. A relationship between high feeding motivation and aggression has been previously reported in some other transgenic species showing enhanced growth. This study aims to elucidate whether the hunger observed in asip1-Tg is linked to aggressive behaviour. Dominance and aggressiveness were quantified using dyadic fights and mirror-stimulus tests, in addition to the analysis of basal cortisol levels. The results indicate that asip1-Tg are less aggressive than WT zebrafish in both dyadic fights and mirror-stimulus tests.

The multifunctional human ocular melanocortin system.

Immune privilege in the eye involves physical barriers, immune regulation and secreted proteins that together limit the damaging effects of intraocular immune responses and inflammation. The neuropeptide alpha-melanocyte stimulating hormone (α-MSH) normally circulates in the aqueous humour of the anterior chamber and the vitreous fluid, secreted by iris and ciliary epithelium, and retinal pigment epithelium (RPE). α-MSH plays an important role in maintaining ocular immune privilege by helping the development of suppressor immune cells and by activating regulatory T-cells. α-MSH functions by binding to and activating melanocortin receptors (MC1R to MC5R) and receptor accessory proteins (MRAPs) that work in concert with antagonists, otherwise known as the melanocortin system. As well as controlling immune responses and inflammation, a broad range of biological functions is increasingly recognised to be orchestrated by the melanocortin system within ocular tissues. This includes maintaining corneal transparency and immune privilege by limiting corneal (lymph)angiogenesis, sustaining corneal epithelial integrity, protecting corneal endothelium and potentially enhancing corneal graft survival, regulating aqueous tear secretion with implications for dry eye disease, facilitating retinal homeostasis via maintaining blood-retinal barriers, providing neuroprotection in the retina, and controlling abnormal new vessel growth in the choroid and retina. The role of melanocortin signalling in uveal melanocyte melanogenesis however remains unclear compared to its established role in skin melanogenesis. The early application of a melanocortin agonist to downregulate systemic inflammation used adrenocorticotropic hormone (ACTH)-based repository cortisone injection (RCI), but adverse side effects including hypertension, edema, and weight gain, related to increased adrenal gland corticosteroid production, impacted clinical uptake. Compared to ACTH, melanocortin peptides that target MC1R, MC3R, MC4R and/or MC5R, but not adrenal gland MC2R, induce minimal corticosteroid production with fewer adverse systemic effects. Pharmacological advances in synthesising MCR-specific targeted peptides provide further opportunities for treating ocular (and systemic) inflammatory diseases. Following from these observations and a renewed clinical and pharmacological interest in the diverse biological roles of the melanocortin system, this review highlights the physiological and disease-related involvement of this system within human eye tissues. We also review the emerging benefits and versatility of melanocortin receptor targeted peptides as non-steroidal alternatives for inflammatory eye diseases such as non-infectious uveitis and dry eye disease, and translational applications in promoting ocular homeostasis, for example, in corneal transplantation and diabetic retinopathy.

Defining Predictors of Weight Loss Response to Lorcaserin.

CONTEXT: Individual responses to weight loss (WL) medications vary widely and prediction of response remains elusive. OBJECTIVE: We investigated biomarkers associated with use of lorcaserin (LOR), a 5HT2cR agonist that targets proopiomelanocortin (POMC) neurons that regulate energy and glucose homeostasis, to identify predictors of clinical efficacy. METHODS: Thirty individuals with obesity were treated with 7 days of placebo and LOR in a randomized crossover study. Nineteen participants continued on LOR for 6 months. Cerebrospinal fluid (CSF) POMC peptide measurements were used to identify potential biomarkers that predict WL. Insulin, leptin, and food intake during a meal were also studied. RESULTS: LOR induced a significant decrease in CSF levels of the POMC prohormone and an increase in its processed peptide ß-endorphin after 7 days; ß-endorphin/POMC increased by 30% (P < .001). This was accompanied by a substantial decrease in insulin, glucose, and homeostasis model assessment of insulin resistance before WL. Changes in CSF POMC peptides persisted after WL (6.9%) at 6 months that were distinct from prior reports after diet alone. Changes in POMC, food intake, or other hormones did not predict WL. However, baseline CSF POMC correlated negatively with WL (P = .07) and a cutoff level of CSF POMC was identified that predicted more than 10% WL. CONCLUSION: Our results provide evidence that LOR affects the brain melanocortin system in humans and that effectiveness is increased in individuals with lower melanocortin activity. Furthermore, early changes in CSF POMC parallel WL-independent improvements in glycemic indexes. Thus, assessment of melanocortin activity could provide a way to personalize pharmacotherapy of obesity with 5HT2cR agonists.

miRNA-383 and miRNA-384 Suppress Proopiomelanocortin Gene Expression in the Hypothalamus: Effects of Early Life Ethanol Exposure.

INTRODUCTION: Early life ethanol exposure is known to program hypothalamic proopiomelanocortin (POMC) neurons to express a reduced level of POMC and its control of stress axis functions throughout the life span. In this study, we tested whether miRNAs contribute to the ethanol-induced suppression of Pomc gene expression during the developmental period. METHODS: In in vivo studies, POMC-EGFP male mice were fed with 2.5 g/kg ethanol using milk formula (AF), pair-fed isocaloric milk formula, or left in the litter during postnatal days (PNDs) 2-6. In in vitro studies, mHypoA-POMC/GFP cells were treated with ethanol (50 mM) for a 24-h period. Hypothalamic tissues or cell extracts were used for measurement of miRNAs and POMC mRNA. RESULTS: Determination of genome-wide microRNA expression profile identified 40 miRNAs significantly altered in hypothalamic tissues of AF mice. In silico analysis further identified miRNA-383, -384, and -488 have putative binding sites at the POMC 3'UTR. However, only miR-383 and miR-384 are identified to be responsive to ethanol. Administration of miR-383 or -384 inhibitor oligos suppressed ethanol-stimulated miR-383 or -384 expression and restored Pomc mRNA and protein expression in AF mice. mHypoA-POMC/GFP cells when treated with ethanol showed elevated levels of miR-383 and miR-384 and reduced level of Pomc mRNA. Treatment with miR-383 or -384 mimic oligos reduced the level of Pomc mRNA, while treatment with miR-383 or -384 inhibitor oligos increased the level of Pomc mRNA. Reporter assay further confirms the binding specificity of miR-383 and miR-384 to Pomc 3'UTR. CONCLUSION: These data suggest that miR-383 and miR-384 suppress Pomc gene expression and may contribute to the ethanol-induced alteration of the stress axis functions.

Proopiomelanocortin projections to the nucleus accumbens modulate acquisition and maintenance of operant palatable pellet administration in mice.

Obesity is a crisis in the United States, producing many co-morbid diseases that can drastically decrease quality of life. While diet is a major focus for therapeutic intervention, the need to understand underlying appetitive neurocircuitry persists. Proopiomelanocortin (POMC) peptides are well-known for their anorexigenic activity, but also mediate reward and learning. The nucleus accumbens (NAcc) is best known for its role in reward-based learning, but the contribution of POMC projections to NAcc on feeding are controversial since the two major POMC-derived peptides (ß-endorphin and α-MSH) have opposite effects on food intake. Our objective was to determine the effect of stimulating POMC projections in the NAcc on acquisition and maintenance of operant self-administration of a palatable food. Adult POMCCre mice were microinjected into the NAcc with a Cre-dependent retrograde adeno-associated viral vector expressing Gq Designer Receptors Exclusively Activated by Designer Drugs (DREADDs). Mice were trained to self-administer palatable 20-mg pellets in daily operant sessions. Acquisition of self-administration (fixed ratio 30) and baseline self-administration were measured in daily sessions, with mice receiving injections of either JHU37152 (DREADD agonist) or saline (i.p.) 15 min prior to the sessions. POMC neuron stimulation (JHU injection) before training sessions produced a significant increase in rate of acquisition and accuracy compared to the saline treated group, with no significant effect on rewards earned. Removal of POMC neuron stimulation before sessions initially reduced consumption with a gradual increase in responding for reinforcer over 3 days of saline injections. Reinstatement of POMC neuron stimulation (JHU) before the session resulted in a significant decrease in responding and rewards earned. These results suggest a complex role of POMC peptides within the NAcc that increase reward learning for a novel palatable food while decreasing consumption of the reinforcer following experience with it.

The Association of the Hypothalamic-Pituitary-Adrenal Axis with Appetite Regulation in Children with Fetal Alcohol Spectrum Disorders (FASDs).

Prenatal alcohol exposure causes growth impairment and a wide range of developmental, physical, and cognitive disorders in children, collectively referred to as fetal alcohol spectrum disorders (FASDs). In the course of FASDs, abnormalities can also affect eating behavior and nutritional status, but these problems have received little attention. Therefore, the aim of our study was to determine the levels of hormones involved in the action of the hypothalamic-pituitary-adrenal axis: proopiomelanocortin (POMC), cortisol, and adrenocorticotropic hormone (ACTH), in the serum of patients with FASDs. To our knowledge, none of these hormones studied have yet been evaluated in FASDs to date. We investigated 62 FASD patients and 23 healthy controls by applying an enzyme-linked immunosorbent method (ELISA). Fasting POMC levels were significantly lower in patients with FASDs (10.97 vs. 18,57 ng/mL, p = 0.039) compared to controls. However, there were no differences in cortisol concentrations. Additionally, the sex and subgroup status (fetal alcohol syndrome (FAS), neurobehavioral disorder associated with prenatal alcohol exposure (ND-PAE), and FASD risk) did not affect hormone levels. POMC was positively correlated with some clinical parameters such as age, BMI percentile, carbohydrate biomarkers, and ACTH. A positive correlation was observed between ACTH and cortisol levels, as well as ACTH and cholesterol levels. Data analysis showed no HPA axis abnormalities in the form of elevated serum cortisol and ACTH levels. Differences in POMC concentration may indicate the involvement and/or impairment of central nervous system structures in hormonal alterations in FASD individuals, caused by prenatal alcohol exposure. Hormonal dysregulation in FASDs can contribute to reduced growth and development, as well as many other disturbed processes, including neurological/neurodevelopmental dysfunctions. Further insightful studies involving a larger group of patients are needed to determine the potential impact of the measured hormones.