Antonio Giordano

Noradrenergic parenchymal nerve fiber branching after cold acclimatisation correlates with brown adipocyte density in mouse adipose organ

The mammalian adipose organ is composed of subcutaneous and visceral depots containing white and brown adipocytes. Cold acclimatisation induces an increase in the brown component without affecting the overall number of adipocytes; this form of plasticity is associated to obesity and diabetes resistance in experimental models. Cold activates the drive of the sympathetic nervous system to the adipose organ, where the vast majority of nerve fibers are in fact noradrenergic. However, it is unclear whether and how such fibers are involved in the plastic changes of the adipose organ. We thus conducted a systematic study of the distribution and number of sympathetic noradrenergic nerve fibers in the adipose organ of mice kept at different environmental temperatures. Adult Sv129 female mice were kept at 28 °C or 6 °C for 10 days. The density of tyrosine hydroxylase (noradrenergic)‐positive nerve fibers (no. of fibers per 100 adipocytes) was calculated in the subcutaneous and visceral depots of the adipose organ, and a correlation was sought between fiber density and proportion of brown adipocytes. Tyrosine hydroxylase‐positive parenchymal fibers were detected in all subcutaneous and visceral depots among white as well as brown adipocytes, the mediastinal depot displaying the densest innervation. Cold acclimatisation induced a threefold increase in the total number of TH fibers in the whole organ. The proportion of brown adipocytes positively correlated with noradrenergic fiber density in the organ. Taken together, these data suggest that cold acclimatisation induces noradrenergic fiber branching in the adipose organ of adult mice, and that such changes may be a precondition for its plastic transformation into a brown phenotype.

Leptin increases axonal growth cone size in developing mouse cortical neurons by convergent signals inactivating glycogen synthase kinase-3β

We examined the effects of the adipose hormone leptin on the development of mouse cortical neurons. Treatment of neonatal and adult mice with intraperitoneal leptin (5 mg/kg) induced extracellular signal-regulated kinase (ERK) 1/2 phosphorylation in pyriform and entorhinal cortex neurons. Stimulation of cultured embryonic cortical neurons with leptin evoked Janus kinase 2 and ERK1/2 phosphorylation and activated the downstream effector 90-kDa ribosomal protein S6 kinase. Moreover, leptin elicited the phosphorylation of the phosphatidylinositol 3-kinase effector Akt and evoked Ser-9 phosphorylation of glycogen synthase kinase-3β (GSK3β), an event inactivating this kinase. Leptin-mediated GSK3β phosphorylation was prevented by the MEK/ERK inhibitor PD98059, the phosphatidylinositol 3-kinase inhibitor LY294002, or the protein kinase C inhibitor GF109203X. Exposure of cortical neurons to leptin also induced Ser-41 phosphorylation of the neuronal growth-associated protein GAP-43, an effect prevented by LY294002 and GF109203X but not by PD98059. Ser-41-GAP-43 phosphorylation is usually high in expanding axonal growth cones. Neurons exposed to 100 ng/ml leptin for 72 h displayed reduced rate of growth cone collapse, a shift of growth cone size distribution toward higher values, and a 4-fold increase in mean growth cone surface area compared with control cultures. The leptin-induced growth cone spreading was hampered in cortical neurons from Leprdb/db mice lacking functional leptin receptors; it was associated with localized Ser-9-GSK3β phosphorylation and mimicked by the GSK3β inhibitor SB216763. At concentrations preventing GSK3β phosphorylation, PD98059, LY294002, or GF109203X reversed the leptin-induced growth cone surface enlargement. We concluded that the leptin-mediated regulation of growth cone morphogenesis in cortical neurons relies on upstream regulators of GSK3β activity.

Leptin Is Induced in the Ischemic Cerebral Cortex and Exerts Neuroprotection Through NF-κB/c-Rel–Dependent Transcription

Background and Purpose— Leptin is an adipose hormone endowed with angiopoietic, neurotrophic, and neuroprotective properties. We tested the hypothesis that leptin might act as an endogenous mediator of recovery after ischemic stroke and investigated whether nuclear transcription factors &kgr;B activation is involved in leptin-mediated neuroprotection. Methods— The antiapoptotic effects of leptin were evaluated in cultured mouse cortical neurons from wild-type or NF-&kgr;B/c-Rel−/− mice exposed to oxygen–glucose deprivation. Wild-type, c-Rel−/− and leptin-deficient ob/ob mice were subjected to permanent middle cerebral artery occlusion. Leptin production was measured in brains from wild-type mice with quantitative reverse transcriptase–polymerase chain reaction and immunostaining. Mice received a leptin bolus (20 μg/g) intraperitoneally at the onset of ischemia. Results— Leptin treatment activated the nuclear translocation of nuclear transcription factors &kgr;B dimers containing the c-Rel subunit, induced the expression of the antiapoptotic c-Rel target gene Bcl-xL in both control and oxygen–glucose deprivation conditions, and counteracted the oxygen–glucose deprivation-mediated apoptotic death of cultured cortical neurons. Leptin-mediated Bcl-xL induction and neuroprotection against oxygen–glucose deprivation were hampered in cortical neurons from c-Rel−/− mice. Leptin mRNA was induced and the protein was detectable in microglia/macrophage cells from the ischemic penumbra of wild-type mice subjected to permanent middle cerebral artery occlusion. Ob/ob mice were more susceptible than wild-type mice to the permanent middle cerebral artery occlusion injury. Leptin injection significantly reduced the permanent middle cerebral artery occlusion-mediated cortical damage in wild-type and ob/ob mice, but not in c-Rel−/− mice. Conclusions— Leptin acts as an endogenous mediator of neuroprotection during cerebral ischemia. Exogenous leptin administration protects against ischemic neuronal injury in vitro and in vivo in a c-Rel-dependent manner.

Regional-dependent Increase of Sympathetic Innervation in Rat White Adipose Tissue during Prolonged Fasting

White adipose tissue (WAT) is innervated by the sympathetic nervous system. A role for WAT sympathetic noradrenergic nerves in lipid mobilization has been suggested. To gain insight into the involvement of nerve activity in the delipidation process, WAT nerves were investigated in rat retroperitoneal and epididymal depots after prolonged fasting. A significant increase in tyrosine hydroxylase (TH) content was found in epididymal and, especially, retroperitoneal WAT by Western blotting. Accordingly, an increased immunoreactivity for TH was detected by immunohistochemistry in epididymal and, especially, retroperitoneal vascular and parenchymal noradrenergic nerves. Neuropeptide Y (NPY)-containing nerves were found around arteries and in the parenchyma. Double-staining experiments and confocal microscopy showed that most perivascular and some parenchymal noradrenergic nerves also contained NPY. Detection of protein gene product (PGP) 9.5, a general marker of peripheral nerves, by Western blotting and PGP 9.5-TH by double-staining experiments showed significantly increased noradrenergic nerve density in fasted retroperitoneal, but not epididymal depots, suggesting that formation of new nerves takes place in retroperitoneal WAT in fasting conditions. On the whole, these data confirm the important role of sympathetic noradrenergic nerves in WAT lipid mobilization during fasting but also raise questions about the physiological role of regional-dependent nerve adjustments and their functional significance in relation to white adipocyte secretory products.

CL316,243 and Cold Stress Induce Heterogeneous Expression of UCP1 mRNA and Protein in Rodent Brown Adipocytes

Uncoupling protein 1 (UCP1), the mammalian thermogenic mitochondrial protein, is found only in brown adipocytes, but its expression by immunohistochemistry is not homogeneous. Here we present evidence that the non-homogeneous pattern of immunostaining for UCP1 (referred to as the “Harlequin phenomenon”) is particularly evident after acute and chronic cold (4C) stimulus and after administration of a specific β3-adrenoceptor agonist (CL316,243). Accordingly, mRNA in situ expression confirmed the UCP1 non-homogeneous pattern of gene activation under conditions of adrenergic stimulus. Furthermore, morphometric analysis of immunogold-stained thin sections showed that UCP1-gold particle density was different among neighboring brown adipocytes with mitochondria of the same size and density. When the adrenergic stimulus was reduced in warm-acclimated animals (28C), UCP1 protein and mRNA expression was reduced and consequently the Harlequin phenomenon was barely visible. These data suggest the existence of an alternative and controlled functional recruitment of brown adipocytes in acute adrenergically stressed animals, possibly to avoid heat and metabolic damage in thermogenically active cells. Of note, the heat shock protein heme oxygenase 1 (HO1) is heterogeneously expressed in adrenergically stimulated brown adipose tissue and, specifically, cells expressing strong immunoreactivity for UCP1 also strongly express HO1.

Presence and Distribution of Cholinergic Nerves in Rat Mediastinal Brown Adipose Tissue

Brown adipose tissue (BAT) is richly provided with sympathetic noradrenergic nerves but is believed to lack a parasympathetic nerve supply. Acetylcholine is the predominant transmitter of postganglionic parasympathetic nerves. The vesicular acetylcholine transporter (VAChT) resides in synaptic vesicles of cholinergic nerve terminals and is used as a marker for peripheral cholinergic nerves. We sought cholinergic nerves in rat BAT using VAChT immunohistochemistry (IHC) on cryosections of interscapular, cervical, mediastinal, and perirenal depots. Mediastinal BAT was the sole depot provided with putative parasympathetic perivascular and parenchymal cholinergic nerves. The absence of vasoactive intestinal peptide-positive nerves suggested their nature as pure cholinergic fibers. By confocal microscopy, both cholinergic and noradrenergic nerves were detected in mediastinal BAT. Cold exposure and fasting led to increased density of VAChT-positive fibers and of noradrenergic sympathetic nerves at morphometry. The unexpected double innervation of mediastinal BAT may explain the inhibitory influence on thermogenesis observed after systemic injection of muscarinic antagonists in rats, and raises questions about the physiological role of its cholinergic nerve supply. (J Histochem Cytochem 52:923–930, 2004)

Leptin-dependent STAT3 phosphorylation in postnatal mouse hypothalamus.

Leptin, a hormone produced by adipose tissue, reduces food intake and boosts energy expenditure via activation of the JAK2-STAT3 signalling pathway in adult mammal hypothalamic neurons. It is found in blood early after birth, peaking around postnatal day (P) 10. The hypothalamus of neonatal mice administered intraperitoneal leptin (3 mg/kg of body weight) was investigated for phospho-STAT3-positive cells to gain insights into the timing of maturation of the leptin signal transduction system. Leptin responsiveness was first detected in arcuate nucleus, where it was faint at P1 and evident from P5. It was then identified in medial preoptic area, anterior hypothalamus, retrochiasmatic area, dorsomedial nucleus and premammillary nucleus from P7, and in ventromedial nucleus and lateral hypothalamus from P11. From P13 onwards, hypothalamic P-STAT3 staining was indistinguishable from that of adult mice. Significant hypothalamic STAT3 activation was also detected by Western blotting at P11 and P15. The level of activation seen in adults was comparable to that observed at P15 although, remarkably, leptin-induced feeding reduction is observed only after the fourth postnatal week. Neuronal and glial markers and double-labelling immunohistochemistry showed that leptin-stimulated hypothalamic cells that had already reached their final position in a given area or nucleus were neurons; however, leptin responsiveness preceded positivity for the neuronal markers, suggesting a not fully differentiated status. Interestingly, leptin also increased P-STAT3 and c-Fos immunoreactivity in a distinctive and transient (from P5 to P13) cell population found in the dorsal part of the third ventricle and in subependymal position. These cells did not express mature or immature neuronal or glial markers. Their ultrastructural appearance, though suggestive of differentiating cells, was not conclusive for a specific phenotype.

Adipose organ nerves revealed by immunohistochemistry.

Brown and white adipose tissue have recently gained prominence as key players in obesity and related health problems, such as type-2 diabetes and cardiovascular disease. Brown adipose tissue-dependent nonshivering thermogenesis significantly affects the bodys energy balance. Originally considered as a passive store of lipids, white adipose tissue has recently been found to secrete a number of hormones and cytokines and to be thus involved in the control of body metabolism and energy balance at multiple sites. These findings have renewed the interest in adipose organ biology, including its innervation by the autonomic nervous system and sensory nerves. Here, we describe our protocols for detecting different types of adipose tissue nerves by light microscopy using peroxidase immunostaining and by laser scanning confocal microscopy using immunofluorescence. With these techniques, the presence, distribution, and colocalization of autonomic and sensory nerves can be effectively investigated in subcutaneous and visceral adipose depots of normal and obese animals.

Molecular aspects of adipoepithelial transdifferentiation in mouse mammary gland.

The circular, reversible conversion of the mammary gland during pregnancy and involution is a paradigm of physiological tissue plasticity. The two most prominent cell types in mammary gland, adipocytes and epithelial cells, interact in an orchestrated way to coordinate this process. Previously, we showed that this conversion is at least partly achieved by reciprocal transdifferentiation between mammary adipocytes and lobulo‐alveolar epithelial cells. Here, we aim to shed more light on the regulators of mammary transdifferentiation. Using immunohistochemistry with cell type‐specific lipid droplet‐coating markers (Perilipin1 and 2), we show that cells with an intermediate adipoepithelial phenotype exist during and after pregnancy. Nuclei of cells with similar transitional structural characteristics are highly positive for Elf5, a master regulator of alveologenesis. In cultured adipocytes, we could show that transient and stable ectopic expression of Elf5 induces expression of the milk component whey acidic protein, although the general adipocyte phenotype is not affected suggesting that additional pioneering factors are necessary. Furthermore, the lack of transdifferentiation of adipocytes during pregnancy after clearing of the epithelial compartment indicates that transdifferentiation signals must emanate from the epithelial part. To explore candidate genes potentially involved in the transdifferentiation process, we devised a high‐throughput gene expression study to compare cleared mammary fat pads with developing, contralateral controls at several time points during pregnancy. Incorporation of bioinformatic predictions of secretory proteins provides new insights into possible paracrine signaling pathways and downstream transdifferentiation factors. We discuss a potential role for osteopontin (secreted phosphoprotein 1 [Spp1]) signaling through integrins to induce adipoepithelial transdifferentiation. Stem Cells 2014;32:2756–2766

Activation of Hypocretin-1/Orexin-A Neurons Projecting to the Bed Nucleus of the Stria Terminalis and Paraventricular Nucleus Is Critical for Reinstatement of Alcohol Seeking by Neuropeptide S

BACKGROUND Environmental conditioning is a major trigger for relapse in abstinent addicts. We showed that activation of the neuropeptide S (NPS) system exacerbates reinstatement vulnerability to cocaine and alcohol via stimulation of the hypocretin-1/orexin-A (Hcrt-1/Ox-A) system. METHODS Combining pharmacologic manipulations with immunohistochemistry techniques, we sought to determine how NPS and Hcrt-1/Ox-A systems interact to modulate reinstatement of alcohol seeking in rats. RESULTS Intrahypothalamic injection of NPS facilitated discriminative cue-induced reinstatement of alcohol seeking. This effect was blocked by the selective Hcrt-1/Ox-A antagonist SB334867 microinjected into the hypothalamic paraventricular nucleus (PVN) or into the bed nucleus of the stria terminalis (BNST) but not into the ventral tegmental area or the locus coeruleus. Combining double labeling and confocal microscopy analyses, we found that NPS-containing axons are in close apposition to hypothalamic Hcrt-1/Ox-A positive neurons, a significant proportion of which express NPS receptors, suggesting a direct interaction between the two systems. Retrograde tracing experiments showed that intra-PVN or intra-BNST red fluorobead unilateral injection labeled bilaterally Hcrt-1/Ox-A somata, suggesting that NPS could recruit two distinct neuronal pathways. Confirming this assumption, intra-BNST or PVN Hcrt-1/Ox-A injection enhanced alcohol seeking similarly to hypothalamic NPS injection but to a lesser degree. CONCLUSIONS Results suggest that the Hcrt-1/Ox-A neurocircuitry mediating the facilitation of cue-induced reinstatement by NPS involves structures critically involved in stress regulation such as the PVN and the BNST. These findings open to the tempting hypothesis of a role of the NPS system in modulating the interactions between stress and environmental conditioning factors in drug relapse.