Genetic and pharmacologic blockade of central melanocortin signaling attenuates cardiac cachexia in rodent models of heart failure

The central melanocortin system plays a key role in the regulation of food intake and energy homeostasis. We investigated whether genetic or pharmacologic blockade of central melanocortin signaling attenuates cardiac cachexia in mice and rats with heart failure. Permanent ligation of the left coronary artery (myocardial infarction (MI)) or sham operation was performed in wild-type (WT) or melanocortin-4 receptor (MC4R) knockout mice. Eight weeks after surgery, WT-Sham mice had significant increases in lean body mass (LBM; P<0·05) and fat mass (P<0·05), whereas WT-MI did not gain significant amounts of LBM or fat mass. Resting basal metabolic rate (BMR) was significantly lower in WT-Sham mice compared to WT-MI mice (P<0·001). In contrast, both MC4-Sham and MC4-MI mice gained significant amounts of LBM (P<0·05) and fat mass (P<0·05) over the study period. There was no significant difference in the BMR between MC4-Sham and MC4-MI mice. In the second experiment, rats received aortic bands or sham operations, and after recovery received i.c.v. injections of either artificial cerebrospinal fluid (aCSF) or the melanocortin antagonist agouti-related protein (AGRP) for 2 weeks. Banded rats receiving AGRP gained significant amount of LBM (P<0·05) and fat mass (P<0·05) over the treatment period, whereas banded rats receiving aCSF did not gain significant amounts of LBM or fat mass. These results demonstrated that genetic and pharmacologic blockade of melanocortin signaling attenuated the metabolic manifestations of cardiac cachexia in murine and rat models of heart failure

Perineuronal Net Formation and the Critical Period for Neuronal Maturation in the Hypothalamic Arcuate Nucleus

In leptin-deficient ob/ob mice, obesity and diabetes are associated with abnormal development of neurocircuits in the hypothalamic arcuate nucleus (ARC)1, a critical brain area for energy and glucose homoeostasis2,3. Because this developmental defect can be remedied by systemic leptin administration, but only if given before postnatal day 28, a critical period for leptin-dependent development of ARC neurocircuits has been proposed4. In other brain areas, critical-period closure coincides with the appearance of perineuronal nets (PNNs), extracellular matrix specializations that restrict the plasticity of neurons that they enmesh5. Here we report that in humans and rodents, subsets of neurons in the mediobasal aspect of the ARC are enmeshed in PNN-like structures. In mice, these neurons are densely packed into a continuous ring that encircles the junction of the ARC and median eminence, which facilitates exposure of ARC neurons to the circulation. Most of the enmeshed neurons are both γ-aminobutyric acid-ergic and leptin-receptor positive, including a majority of Agouti-related-peptide neurons. Postnatal formation of the PNN-like structures coincides precisely with closure of the critical period for maturation of Agouti-related-peptide neurons and is dependent on input from circulating leptin, because postnatal ob/ob mice have reduced ARC PNN-like material that is restored by leptin administration during the critical period. We conclude that neurons crucial to metabolic homoeostasis are enmeshed in PNN-like structures and organized into a densely packed cluster situated circumferentially at the ARC–median eminence junction, where metabolically relevant humoral signals are sensed

Transcriptomic analysis links diverse hypothalamic cell types to fibroblast growth factor 1-induced sustained diabetes remission

n rodent models of type 2 diabetes (T2D), sustained remission of hyperglycemia can be induced by a single intracerebroventricular (icv) injection of fibroblast growth factor 1 (FGF1), and the mediobasal hypothalamus (MBH) was recently implicated as the brain area responsible for this effect. To better understand the cellular response to FGF1 in the MBH, we sequenced >79,000 single-cell transcriptomes from the hypothalamus of diabetic Lepob/ob mice obtained on Days 1 and 5 after icv injection of either FGF1 or vehicle. A wide range of transcriptional responses to FGF1 was observed across diverse hypothalamic cell types, with glial cell types responding much more robustly than neurons at both time points. Tanycytes and ependymal cells were the most FGF1-responsive cell type at Day 1, but astrocytes and oligodendrocyte lineage cells subsequently became more responsive. Based on histochemical and ultrastructural evidence of enhanced cell-cell interactions between astrocytes and Agrp neurons (key components of the melanocortin system), we performed a series of studies showing that intact melanocortin signaling is required for the sustained antidiabetic action of FGF1. These data collectively suggest that hypothalamic glial cells are leading targets for the effects of FGF1 and that sustained diabetes remission is dependent on intact melanocortin signaling

Cloning and distribution of galanin-like peptide mRNA in the hypothalamus and pituitary of the macaque

Galanin-like peptide (GALP) is a newly discovered hypothalamic neuropeptide, which is regulated by leptin and implicated in the regulation of GnRH secretion in the rodent. We searched the human genome database and determined that the human GALP gene comprises six exons, as has been shown for human galanin. We used rapid amplification of cDNA ends to clone a full-length cDNA (802 bp) of the macaque homologue of GALP and found it to be highly conserved between human and macaque at both the nucleotide (93%) and peptide (94%) levels. Mature GALP is predicted to be 60 amino acids in the macaque as in other species, and the region of GALP (9-21) that shows homology to the N-terminal 13 amino acids of galanin is perfectly conserved. We mapped the distribution of GALP mRNA in the hypothalamus and pituitary of the macaque by in situ hybridization and observed that, as in rodent species, the expression of GALP mRNA is confined to the arcuate nucleus, median eminence, and neurohypophysis. Using double-label in situ hybridization, we found that nearly all (98%) GALP mRNA-expressing cells in the arcuate nucleus also express mRNA for the long form of the leptin receptor. These findings suggest that a leptin-GALP signaling pathway exists in a primate species

Prolonged Adherence of Human Immunodeficiency Virus-Derived Vector Particles to Hematopoietic Target Cells Leads to Secondary Transduction In Vitro and In Vivo

Human immunodeficiency virus type 1-derived lentivirus vectors bearing the vesicular stomatitis virus G (VSV-G) envelope glycoprotein demonstrate a wide host range and can stably transduce quiescent hematopoietic stem cells. In light of concerns about biosafety and potential germ line transmission, they have been used predominantly for ex vivo strategies, thought to ensure the removal of excess surface-bound particles and prevent in vivo dissemination. Studies presented here instead reveal prolonged particle adherence after ex vivo exposure, despite serial wash procedures, with subsequent transduction of secondary target cells in direct and transwell cocultures. We explored the critical parameters affecting particle retention and transfer and show that attachment to the cell surface selectively protects virus particles from serum complement-mediated inactivation. Moreover, studies with nonmyeloablated murine recipients show that transplantation of vector-exposed, washed hematopoietic cells results in systemic dissemination of functional VSV-G/lentivector particles. We demonstrate genetic marking by inadvertent transfer of vector particles and prolonged expression of transgene product in recipient tissues. Our findings have implications for biosafety, vector design, and cell biology research

Galanin-like peptide as a possible link between metabolism and reproduction in the macaque

Galanin-like peptide (GALP) is a hypothalamic neuropeptide that has been implicated in the control of feeding, metabolism, and reproduction. The goal of this study was to examine the effects of central infusions of GALP on GnRH and LH secretion and to identify physiological factors that influence the expression of GALP mRNA in the brain of a primate species. Infusions of GALP into the lateral cerebroventricle of the macaque caused a significant increase in LH secretion, which was blocked by administration of the GnRH receptor antagonist acyline. However, the expression of GALP mRNA in the arcuate nucleus, as determined by in situ hybridization, was not regulated by either estradiol or progesterone. Compared with feeding ad libitum, fasting for 48 h produced a significant reduction in the hypothalamic expression of GALP mRNA. GALP neurons were found to express both neuropeptide Y Y1 receptor and serotonin 2C receptor by double-label in situ hybridization. Taken together, these results suggest that GALP neurons play a role of integrating metabolic signals, which are relayed to circuits controlling GnRH release in the macaque

Perineuronal Net Formation during the Critical Period for Neuronal Maturation in the Hypothalamic Arcuate Nucleus

In leptin-deficient mice, obesity and diabetes are associated with abnormal development of neurocircuits in the hypothalamic arcuate nucleus (ARC), a critical brain area for energy and glucose homeostasis. As this developmental defect can be remedied by systemic leptin administration, but only if given before postnatal day 28, a critical period (CP) for leptin-dependent development of ARC neurocircuits has been proposed. In other brain areas, CP closure coincides with the appearance of perineuronal nets (PNNs), extracellular matrix specializations that restrict the plasticity of neurons that they enmesh. Here we report that in humans as well as rodents, subsets of neurons in the mediobasal aspect of the ARC are enmeshed by PNN-like structures. In mice, these neurons are densely-packed into a continuous ring that encircles the junction of the ARC and median eminence, which facilitates exposure of ARC neurons to the circulation. Most of the enmeshed neurons are both GABAergic and leptin receptor-positive, including a majority of Agrp neurons. Postnatal formation of the PNN-like structures coincides precisely with closure of the CP for Agrp neuron maturation and is dependent on input from circulating leptin, as postnatal mice have reduced ARC PNN-like material that is restored by leptin administration during the CP. We conclude that neurons crucial to metabolic homeostasis are enmeshed by PNN-like structures and organized into a densely packed cluster situated circumferentially at the ARC-ME junction, where metabolically-relevant humoral signals are sensed