The ciliary neurotrophic factor (CNTF) is a neurotrophic factor acting on both neurons and glial cells. As it is involved in the maintenance of motor circuits in the postnatal period, possible therapeutic effects of Axokine, an analog of human recombinant CNTF, have been evaluated in patients affected by motor disorders. Despite effects on motor skills were not encouraging, the patients treated with Axokine showed a significant weight loss related to a reduction in appetite. Starting from these observations, many research groups evaluated the effects of CNTF administration in the hypothalamus, the pivotal center of the energy balance regulation in mammals. Collectively, these studies confirmed that CNTF induces satiety, reduction of food intake and increase of energy expenditure through an action on the hypothalamic neuronal circuits that are responsive to leptin, insulin and other circulating factors. As little is known about the presence and distribution of CNTF in the hypothalamus, in normal or pathological conditions, here we aimed to assess in mice 1) whether CNTF is expressed in the hypothalamus, 2) which cell types produce it, and 3) whether the hypothalamic expression of CNTF is modulated by different nutritional conditions. The results from RT-PCR and Western blotting analyses suggest that CNTF is constitutively expressed in the hypothalamus of mice kept in standard conditions. By immunohistochemistry, CNTF is produced by some subependymal astrocytes and ependymal cells. The tanycytes of the tuberal and mammillary hypothalamus, where the neuronal populations involved in the regulation of feeding behavior are located, were strongly positive for CNTF. To assess a possible role of endogenous CNTF in the regulation of energy balance, its expression was evaluated in obese mice fed with an high-fat diet (HFD) and, conversely, in lean mice, kept in conditions of calorie restriction (60% of normal daily caloric intake). The results showed that obesity induced by a HFD is characterized by a significant increase of the expression of CNTF in the tanycytes of the tuberal region of the hypothalamus, whereas the caloric restriction determines the opposite effect. To confirm the involvement of CNTF in the regulation of energy balance in the hypothalamus, we then analyzed the expression of its specific receptor. RT-PCR and Western blotting revealed that, similar to the ligand, obesity induced by a HFD caused a significant increase in the hypothalamic expression of CNTF receptor; the opposite was true for the calorie restriction condition. Given the unavailability of specific antibodies against the CNTF receptor, CNTF-responsive cells were detected by P-STAT3 immunohistochemistry in mice acutely treated with CNTF. Ependymal cells, importantly tanycytes, were the main hypothalamic cells responding to CNTF. In agreement with molecular data, in obese HFD mice the responsiveness of the ependyma to CNTF increased, whereas it decreased in calorie restricted mice. In conclusion, the CNTF is produced by the ependymal cells of the third ventricle, in particular by the tanycytes of the tuberal and mammillary regions of the hypothalamus. It represents a new satiety factor involved in the pathophysiological regulation of the energy balance. The pharmacological modulation of its expression and/or of its specific receptor could be an innovative approach to the treatment of human obesity.
Il fattore neurotrofico ciliare (ciliary neurotrophic factor, CNTF) è un fattore neurotrofico attivo sia su neuroni che su cellule gliali. Poiché in epoca postnatale il CNTF mantiene il trofismo dei circuiti motori, eventuali effetti terapeutici di Axokine, analogo ricombinante del CNTF umano, sono stati valutati su pazienti affetti da patologie motorie. Mentre gli effetti sulle abilità motorie si sono rivelati scarsi, i pazienti trattati con Axokine hanno presentato un significativo calo ponderale legato ad una riduzione dell’appetito. Numerosi gruppi di ricerca hanno quindi valutato l’azione del CNTF nell’ipotalamo, sede dei circuiti neuronali che nei mammiferi regolano il bilancio energetico. Nel complesso, il CNTF esogeno determina sazietà, riduzione dell’introito di cibo e aumento della spesa energetica, modulando funzione e struttura dei circuiti neuronali ipotalamici sensibili alla leptina, insulina ed altri fattori circolanti. Nulla è noto circa la presenza e la distribuzione del CNTF nell’ipotalamo, in condizioni normali o patologiche, e in questo studio condotto su topi, ci siamo proposti di valutare 1) se il CNTF è espresso nell’ipotalamo, 2) quali tipi cellulari lo producono e 3) se l’espressione ipotalamica del CNTF è modulata da differenti condizioni nutritive. I dati di RT-PCR e Western blotting mostrano che il CNTF è costitutivamente espresso nell’ipotalamo di topi tenuti in condizioni standard. L’analisi immunoistochimica mostra che il CNTF è prodotto da alcune cellule gliali in sede subependimale e dall’ependima del terzo ventricolo, dove sono intensamente positivi i taniciti dell’ipotalamo tuberale e mammillare, regioni ipotalamiche che contengono le popolazioni neuronali che regolano il comportamento alimentare. Al fine di verificare un possibile ruolo del CNTF endogeno nella regolazione del bilancio energetico, l’espressione del CNTF è stata studiata in topi resi obesi da dieta ad alto contenuto lipidico e, all’opposto, in topi magri, tenuti in condizione di restrizione calorica (60% del normale introito calorico giornaliero). I risultati hanno evidenziato che l’obesità da dieta iperlipidica determina un significativo incremento dell’espressione di CNTF nei taniciti delle regioni tuberale e mammillare dell’ipotalamo, mentre la restrizione calorica ha un effetto opposto. Per confermare il coinvolgimento del CNTF nella regolazione del bilancio energetico a livello ipotalamico, abbiamo quindi studiato l’espressione del recettore specifico. I dati di RT-PCR e Western blotting hanno mostrato che, parallelamente a quanto osservato per il ligando, l’obesità da dieta iperlipidica determina un aumento significativo dell’espressione ipotalamica del recettore per il CNTF, mentre la restrizione calorica si associa ad una sua significativa riduzione. Non essendo disponibili anticorpi specifici per il recettore del CNTF, i target cellulari del CNTF sono stati individuati mediante valutazione immunoistochimica dell’attivazione della specifica via di trasduzione (Jak1/2-STAT3 pathway) in topi trattati in acuto con bolo di CNTF. Le principali cellule target del CNTF sono le cellule ependimali, tra cui i taniciti. A conferma dei dati di biologia molecolare, nei topi obesi la responsività di queste cellule al CNTF aumenta, all’opposto nei topi tenuti in condizioni di restrizione calorica si riduce. In conclusione, il CNTF è prodotto dall’ependima del terzo ventricolo, ed in particolare dai taniciti della regione tuberale e mammillare. Esso rappresenta un nuovo fattore di sazietà coinvolto nella regolazione fisiopatologica dei circuiti ipotalamici del bilancio energetico. La modulazione farmacologica della sua espressione e/o del recettore specifico potrebbe rappresentare in futuro un approccio al trattamento farmacologico dell’obesità umana.
Ciliary neurotrophic factor in the mouse hypothalamus. A possible role in energy balance homeostasis / Severi, Ilenia. - (2014 Mar 28).
Ciliary neurotrophic factor in the mouse hypothalamus. A possible role in energy balance homeostasis
Severi, Ilenia
2014-03-28
Abstract
The ciliary neurotrophic factor (CNTF) is a neurotrophic factor acting on both neurons and glial cells. As it is involved in the maintenance of motor circuits in the postnatal period, possible therapeutic effects of Axokine, an analog of human recombinant CNTF, have been evaluated in patients affected by motor disorders. Despite effects on motor skills were not encouraging, the patients treated with Axokine showed a significant weight loss related to a reduction in appetite. Starting from these observations, many research groups evaluated the effects of CNTF administration in the hypothalamus, the pivotal center of the energy balance regulation in mammals. Collectively, these studies confirmed that CNTF induces satiety, reduction of food intake and increase of energy expenditure through an action on the hypothalamic neuronal circuits that are responsive to leptin, insulin and other circulating factors. As little is known about the presence and distribution of CNTF in the hypothalamus, in normal or pathological conditions, here we aimed to assess in mice 1) whether CNTF is expressed in the hypothalamus, 2) which cell types produce it, and 3) whether the hypothalamic expression of CNTF is modulated by different nutritional conditions. The results from RT-PCR and Western blotting analyses suggest that CNTF is constitutively expressed in the hypothalamus of mice kept in standard conditions. By immunohistochemistry, CNTF is produced by some subependymal astrocytes and ependymal cells. The tanycytes of the tuberal and mammillary hypothalamus, where the neuronal populations involved in the regulation of feeding behavior are located, were strongly positive for CNTF. To assess a possible role of endogenous CNTF in the regulation of energy balance, its expression was evaluated in obese mice fed with an high-fat diet (HFD) and, conversely, in lean mice, kept in conditions of calorie restriction (60% of normal daily caloric intake). The results showed that obesity induced by a HFD is characterized by a significant increase of the expression of CNTF in the tanycytes of the tuberal region of the hypothalamus, whereas the caloric restriction determines the opposite effect. To confirm the involvement of CNTF in the regulation of energy balance in the hypothalamus, we then analyzed the expression of its specific receptor. RT-PCR and Western blotting revealed that, similar to the ligand, obesity induced by a HFD caused a significant increase in the hypothalamic expression of CNTF receptor; the opposite was true for the calorie restriction condition. Given the unavailability of specific antibodies against the CNTF receptor, CNTF-responsive cells were detected by P-STAT3 immunohistochemistry in mice acutely treated with CNTF. Ependymal cells, importantly tanycytes, were the main hypothalamic cells responding to CNTF. In agreement with molecular data, in obese HFD mice the responsiveness of the ependyma to CNTF increased, whereas it decreased in calorie restricted mice. In conclusion, the CNTF is produced by the ependymal cells of the third ventricle, in particular by the tanycytes of the tuberal and mammillary regions of the hypothalamus. It represents a new satiety factor involved in the pathophysiological regulation of the energy balance. The pharmacological modulation of its expression and/or of its specific receptor could be an innovative approach to the treatment of human obesity.File | Dimensione | Formato | |
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