Evaluation of CSF and plasma biomarkers of brain melanocortin activity in response to caloric restriction in humans

The melanocortin neuronal system, which consists of hypothalamic proopiomelanocortin (POMC) and agouti-related protein (AgRP) neurons, is a leptin target that regulates energy balance and metabolism, but studies in humans are limited by a lack of reliable biomarkers to assess brain melanocortin activity. The objective of this study was to measure the POMC prohormone and its processed peptide, β-endorphin (β-EP), in cerebrospinal fluid (CSF) and AgRP in CSF and plasma after calorie restriction to validate their utility as biomarkers of brain melanocortin activity. CSF and plasma were obtained from 10 lean and obese subjects after fasting (40 h) and refeeding (24 h), and from 8 obese subjects before and after 6 wk of dieting (800 kcal/day) to assess changes in neuropeptide and hormone levels. After fasting, plasma leptin decreased to 35%, and AgRP increased to 153% of baseline. During refeeding, AgRP declined as leptin increased; CSF β-EP increased, but POMC did not change. Relative changes in plasma and CSF leptin were blunted in obese subjects. After dieting, plasma and CSF leptin decreased to 46% and 70% of baseline, CSF POMC and β-EP decreased, and plasma AgRP increased. At baseline, AgRP correlated negatively with insulin and homeostasis model assessment (HOMA-IR), and positively with the Matsuda index. Thus, following chronic calorie restriction, POMC and β-EP declined in CSF, whereas acutely, only β-EP changed. Plasma AgRP, however, increased after both acute and chronic calorie restriction. These results support the use of CSF POMC and plasma AgRP as biomarkers of hypothalamic melanocortin activity and provide evidence linking AgRP to insulin sensitivity

Functional heterogeneity of POMC neurons relies on mTORC1 signaling.

Hypothalamic pro-opiomelanocortin (POMC) neurons are known to trigger satiety. However, these neuronal cells encompass heterogeneous subpopulations that release γ-aminobutyric acid (GABA), glutamate, or both neurotransmitters, whose functions are poorly defined. Using conditional mutagenesis and chemogenetics, we show that blockade of the energy sensor mechanistic target of rapamycin complex 1 (mTORC1) in POMC neurons causes hyperphagia by mimicking a cellular negative energy state. This is associated with decreased POMC-derived anorexigenic α-melanocyte-stimulating hormone and recruitment of POMC/GABAergic neurotransmission, which is restrained by cannabinoid type 1 receptor signaling. Electrophysiology and optogenetic studies further reveal that pharmacological blockade of mTORC1 simultaneously activates POMC/GABAergic neurons and inhibits POMC/glutamatergic ones, implying that the functional specificity of these subpopulations relies on mTORC1 activity. Finally, POMC neurons with different neurotransmitter profiles possess specific molecular signatures and spatial distribution. Altogether, these findings suggest that mTORC1 orchestrates the activity of distinct POMC neurons subpopulations to regulate feeding behavior

Cerebrospinal fluid levels of leptin, proopiomelanocortin, and agouti-related protein in human pregnancy:evidence for leptin resistance

CONTEXT: Leptin suppresses appetite by modulating the expression of hypothalamic neuropeptides including proopiomelanocortin (POMC) and agouti-related peptide (AgRP). Yet during pregnancy, caloric consumption increases despite elevated plasma leptin levels. DESIGN AND PARTICIPANTS: To investigate this paradox, we measured leptin and soluble leptin receptor in plasma and leptin, POMC, and AgRP in cerebrospinal fluid (CSF) from 21 fasting pregnant women before delivery by cesarean section at a university hospital and from 14 fasting nonpregnant women. RESULTS: Prepregnancy body mass index was 24.6 ± 1.1 (se) vs. 31.3 ± 1.3 at term vs. 26.5 ± 1.6 kg/m(2) in controls. Plasma leptin (32.9 ± 4.6 vs. 16.7 ± 3.0 ng/ml) and soluble leptin receptor (30.9 ± 2.3 vs. 22.1 ± 1.4 ng/ml) levels were significantly higher in pregnant women. However, mean CSF leptin did not differ between the two groups (283 ± 34 vs. 311 ± 32 pg/ml), consistent with a relative decrease in leptin transport into CSF during pregnancy. Accordingly, the CSF/plasma leptin percentage was 1.0 ± 0.01% in pregnant subjects vs. 2.1 ± 0.2% in controls (P < 0.0001). Mean CSF AgRP was significantly higher in pregnant subjects (32.3 ± 2.7 vs. 23.5 ± 2.5 pg/ml; P = 0.03). Mean CSF POMC was not significantly different in pregnant subjects (200 ± 13.6 vs. 229 ± 17.3 fmol/ml; P = 0.190). However, the mean AgRP/POMC ratio was significantly higher among pregnant women (P = 0.003), consistent with an overall decrease in melanocortin tone favoring increased food intake during pregnancy. CONCLUSIONS: These data demonstrate that despite peripheral hyperleptinemia, positive energy balance is achieved during pregnancy by a relative decrease in central leptin concentrations and resistance to leptin's effects on target neuropeptides that regulate energy balance

Clinical utility of plasma POMC and AgRP measurements in the differential diagnosis of ACTH-dependent Cushing's syndrome

CONTEXT: Distinguishing between pituitary [Cushing's disease (CD)] and ectopic causes [ectopic ACTH syndrome (EAS)] of ACTH-dependent Cushing's syndrome can be challenging. Inferior petrosal sinus sampling (IPSS) best discriminates between CD and occult EAS but is a specialized procedure that is not widely available. Identifying adjunctive diagnostic tests may prove useful. In EAS, abnormal processing of the ACTH precursor proopiomelanocortin (POMC) and the accumulation of POMC-derived peptides might be expected and abnormal levels of other neuropeptides may be detected. OBJECTIVE: The objective of the study was to evaluate the diagnostic utility of POMC measurements for distinguishing between CD and occult EAS in patients referred for IPSS. Another objective of the study was to evaluate in parallel the diagnostic utility of another neuropeptide, agouti-related protein (AgRP), because we have observed a 10-fold elevation of AgRP in plasma in a patient with EAS from small-cell lung cancer. DESIGN AND PARTICIPANTS: Plasma POMC and AgRP were measured in 38 Cushing's syndrome patients presenting for IPSS, with either no pituitary lesion or a microadenoma on magnetic resonance imaging, and in 38 healthy controls. RESULTS: Twenty-seven of 38 patients had CD; 11 of 38 had EAS. The mean POMC was higher in EAS vs CD [54.5 ± 13.0 (SEM) vs 17.2 ± 1.5 fmol/mL; P < .05]. Mean AgRP was higher in EAS vs CD (280 ± 76 vs 120 ± 16 pg/mL; P = .01). Although there was an overlap in POMC and AgRP levels between the groups, the POMC levels greater than 36 fmol/mL (n = 7) and AgRP levels greater than 280 pg/mL (n = 3) were specific for EAS. When used together, POMC greater than 36 fmol/mL and/or AgRP greater than 280 pg/mL detected 9 of 11 cases of EAS, indicating that elevations in these peptides have a high positive predictive value for occult EAS. CONCLUSIONS: Expanding upon previous observations of high POMC in EAS, this study specifically demonstrates elevated POMC levels can identify occult ectopic tumors. Elevations in AgRP also favor the diagnosis of EAS, suggesting AgRP should be further evaluated as a potential neuroendocrine tumor marker

PC1/3 Deficiency Impacts Pro-opiomelanocortin Processing in Human Embryonic Stem Cell-Derived Hypothalamic Neurons

We recently developed a technique for generating hypothalamic neurons from human pluripotent stem cells. Here, as proof of principle, we examine the use of these cells in modeling of a monogenic form of severe obesity: PCSK1 deficiency. The cognate enzyme, PC1/3, processes many prohormones in neuroendocrine and other tissues. We generated PCSK1 (PC1/3)-deficient human embryonic stem cell (hESC) lines using both short hairpin RNA and CRISPR-Cas9, and investigated pro-opiomelanocortin (POMC) processing using hESC-differentiated hypothalamic neurons. The increased levels of unprocessed POMC and the decreased ratios (relative to POMC) of processed POMC-derived peptides in both PCSK1 knockdown and knockout hESC-derived neurons phenocopied POMC processing reported in PC1/3-null mice and PC1/3-deficient patients. PC1/3 deficiency was associated with increased expression of melanocortin receptors and PRCP (prolylcarboxypeptidase, a catabolic enzyme for α-melanocyte stimulating hormone (αMSH)), and reduced adrenocorticotropic hormone secretion. We conclude that the obesity accompanying PCSK1 deficiency may not be primarily due to αMSH deficiency

Evaluation of CSF and plasma biomarkers of brain melanocortin activity in response to caloric restriction in humans

The melanocortin neuronal system, which consists of hypothalamic proopiomelanocortin (POMC) and agouti-related protein (AgRP) neurons, is a leptin target that regulates energy balance and metabolism, but studies in humans are limited by a lack of reliable biomarkers to assess brain melanocortin activity. The objective of this study was to measure the POMC prohormone and its processed peptide, β-endorphin (β-EP), in cerebrospinal fluid (CSF) and AgRP in CSF and plasma after calorie restriction to validate their utility as biomarkers of brain melanocortin activity. CSF and plasma were obtained from 10 lean and obese subjects after fasting (40 h) and refeeding (24 h), and from 8 obese subjects before and after 6 wk of dieting (800 kcal/day) to assess changes in neuropeptide and hormone levels. After fasting, plasma leptin decreased to 35%, and AgRP increased to 153% of baseline. During refeeding, AgRP declined as leptin increased; CSF β-EP increased, but POMC did not change. Relative changes in plasma and CSF leptin were blunted in obese subjects. After dieting, plasma and CSF leptin decreased to 46% and 70% of baseline, CSF POMC and β-EP decreased, and plasma AgRP increased. At baseline, AgRP correlated negatively with insulin and homeostasis model assessment (HOMA-IR), and positively with the Matsuda index. Thus, following chronic calorie restriction, POMC and β-EP declined in CSF, whereas acutely, only β-EP changed. Plasma AgRP, however, increased after both acute and chronic calorie restriction. These results support the use of CSF POMC and plasma AgRP as biomarkers of hypothalamic melanocortin activity and provide evidence linking AgRP to insulin sensitivity

Partially redundant enhancers cooperatively maintain Mammalian Pomc expression above a critical functional threshold

Cell-specific expression of many genes is conveyed by multiple enhancers, with each individual enhancer controlling a particular expression domain. In contrast, multiple enhancers drive similar expression patterns of some genes involved in embryonic development, suggesting regulatory redundancy. Work in Drosophila has indicated that functionally overlapping enhancers canalize development by buffering gene expression against environmental and genetic disturbances. However, little is known about regulatory redundancy in vertebrates and in genes mainly expressed during adulthood. Here we study nPE1 and nPE2, two phylogenetically conserved mammalian enhancers that drive expression of the proopiomelanocortin gene (Pomc) to the same set of hypothalamic neurons. The simultaneous deletion of both enhancers abolished Pomc expression at all ages and induced a profound metabolic dysfunction including early-onset extreme obesity. Targeted inactivation of either nPE1 or nPE2 led to very low levels of Pomc expression during early embryonic development indicating that both enhancers function synergistically. In adult mice, however, Pomc expression is controlled additively by both enhancers, with nPE1 being responsible for ∼80% and nPE2 for ∼20% of Pomc transcription. Consequently, nPE1 knockout mice exhibit mild obesity whereas nPE2-deficient mice maintain a normal body weight. These results suggest that nPE2-driven Pomc expression is compensated by nPE1 at later stages of development, essentially rescuing the earlier phenotype of nPE2 deficiency. Together, these results reveal that cooperative interactions between the enhancers confer robustness of Pomc expression against gene regulatory disturbances and preclude deleterious metabolic phenotypes caused by Pomc deficiency in adulthood. Thus, our study demonstrates that enhancer redundancy can be used by genes that control adult physiology in mammals and underlines the potential significance of regulatory sequence mutations in common diseases.Fil: Lam, Daniel D.. University of Michigan. Medical School. Department of Molecular and Integrative Physiology; Estados UnidosFil: Silva Junqueira de Souza, Flavio. Consejo Nacional de Investigaciones Científicas y Técnicas. Instituto de Investigaciones en Ingeniería Genética y Biología Molecular; Argentina. Universidad de Buenos Aires. Facultad de Ciencias Exactas y Naturales; ArgentinaFil: Nasif, Sofia. Consejo Nacional de Investigaciones Científicas y Técnicas. Instituto de Investigaciones en Ingeniería Genética y Biología Molecular; ArgentinaFil: Yamashita, Miho. University of Michigan. Medical School. Department of Molecular and Integrative Physiology; Estados UnidosFil: López Leal, Rodrigo. Centro de Estudios Científicos; ChileFil: Otero Corchon, Veronica. University of Michigan. Medical School. Department of Molecular and Integrative Physiology; Estados UnidosFil: Meece, Kana. Columbia University. College of Physicians and Surgeons. Department of Medicine; Estados UnidosFil: Sampath, Harini. Oregon Health & Science University. Center for Research on Occupational and Environmental Toxicology; Estados UnidosFil: Mercer, Aaron J.. University of Michigan. Medical School. Department of Molecular and Integrative Physiology; Estados UnidosFil: Wardlaw, Sharon L.. Columbia University. College of Physicians and Surgeons. Department of Medicine; Estados UnidosFil: Rubinstein, Marcelo. Consejo Nacional de Investigaciones Científicas y Técnicas. Instituto de Investigaciones en Ingeniería Genética y Biología Molecular; Argentina. University of Michigan. Medical School. Department of Molecular and Integrative Physiology; Estados Unidos. Universidad de Buenos Aires. Facultad de Ciencias Exactas y Naturales; ArgentinaFil: Low, Malcolm J.. University of Michigan. Medical School. Department of Molecular and Integrative Physiology; Estados Unido