Cdk1 inactivation terminates mitotic checkpoint surveillance and stabilizes kinetochore attachments in anaphase

Two mechanisms safeguard the bipolar attachment of chromosomes in mitosis. A correction mechanism destabilizes erroneous attachments that do not generate tension across sister kinetochores [1]. In response to unattached kinetochores, the mitotic checkpoint delays anaphase onset by inhibiting the anaphase-promoting complex/cyclosome (APC/CCdc20) [2]. Upon satisfaction of both pathways, the APC/CCdc20 elicits the degradation of securin and cyclin B [3]. This liberates separase triggering sister chromatid disjunction and inactivates cyclin-dependent kinase 1 (Cdk1) causing mitotic exit. How eukaryotic cells avoid the engagement of attachment monitoring mechanisms when sister chromatids split and tension is lost at anaphase is poorly understood [4]. Here we show that Cdk1 inactivation disables mitotic checkpoint surveillance at anaphase onset in human cells. Preventing cyclin B1 proteolysis at the time of sister chromatid disjunction destabilizes kinetochore-microtubule attachments and triggers the engagement of the mitotic checkpoint. As a consequence, mitotic checkpoint proteins accumulate at anaphase kinetochores, the APC/CCdc20 is inhibited, and securin reaccumulates. Conversely, acute pharmacological inhibition of Cdk1 abrogates the engagement and maintenance of the mitotic checkpoint upon microtubule depolymerization. We propose that the simultaneous destruction of securin and cyclin B elicited by the APC/CCdc20 couples chromosome segregation to the dissolution of attachment monitoring mechanisms during mitotic exit

Hypothalamic mTOR signaling mediates the orexigenic action of ghrelin

Current evidence suggests that ghrelin, a stomach derived peptide, exerts its orexigenic action through specific modulation of Sirtuin1 (SIRT1)/p53 and AMP-activated protein kinase (AMPK) pathways, which ultimately increase the expression of agouti-related protein (AgRP) and neuropeptide Y (NPY) in the arcuate nucleus of the hypothalamus (ARC). However, there is a paucity of data about the possible action of ghrelin on alternative metabolic pathways at this level. Here, we demonstrate that ghrelin elicits a marked upregulation of the hypothalamic mammalian target of rapamycin (mTOR) signaling pathway. Of note, central inhibition of mTOR signaling with rapamycin decreased ghrelin's orexigenic action and normalized the mRNA expression of AgRP and NPY, as well as their key downstream transcription factors, namely cAMP response-element binding protein (pCREB) and forkhead box O1 (FoxO1, total and phosphorylated). Taken together, these data indicate that, in addition to previous reported mechanisms, ghrelin also promotes feeding through modulation of hypothalamic mTOR pathway

Neonatal events, such as androgenization and postnatal overfeeding, modify the response to ghrelin

It is currently accepted that ambient, non-genetic factors influence perinatal development and evoke structural and functional changes that may persist throughout life. Overfeeding and androgenization after birth are two of these key factors that could result in "metabolic imprinting" of neuronal circuits early in life and, thereby, increase the body weight homeostatic "set point", stimulate appetite, and result in obesity. Our aim was to determine the influence of these obesogenic factors on the response to ghrelin. We observed the expected orexigenic effect of ghrelin regardless of the nutritional or hormonal manipulations to which the animals were subjected to at early postnatal development and this effect remained intact at later stages of development. In fact, ghrelin responses increased significantly when the animals were subjected to one of the two manipulations, but not when both were combined. An increased response to ghrelin could explain the obese phenotype displayed by individuals with modified perinatal environment.Ministerio de Educacion y CienciaInstituto de Salud Carlos IIIEuropean Community´s Seventh Framework Programm

Neonatal events, such as androgenization and postnatal overfeeding, modify the response to ghrelin

It is currently accepted that ambient, non-genetic factors influence perinatal development and evoke structural and functional changes that may persist throughout life. Overfeeding and androgenization after birth are two of these key factors that could result in “metabolic imprinting” of neuronal circuits early in life and, thereby, increase the body weight homeostatic “set point”, stimulate appetite and result in obesity. Our aim was to determine the influence of these obesogenic factors on the response to ghrelin. We observed the expected orexigenic effect of ghrelin regardless of the nutritional or hormonal manipulations to which the animals were subjected to at early postnatal development and this effect remained intact at later stages of development. In fact, ghrelin responses increased significantly when the animals were subjected to one of the two manipulations, but not when both were combined. An increased response to ghrelin could explain the obese phenotype displayed by individuals with modified perinatal environment.This work has been supported by grants from Ministerio de Educacion y Ciencia (CD: BFU2011-29102) and Centro de Investigación Biomédica en Red (CIBER) de Fisiopatología de la Obesidad y Nutrición (CIBERobn). CIBERobn is an initiative of the Instituto de Salud Carlos III (ISCIII) of Spain which is supported by FEDER funds. The research leading to these results has also received funding from the European Community's Seventh Framework Programme under the following grant: CD: FP7/2007-2013: n° 245009: NeuroFASTS

Hypothalamic mTOR signaling mediates the orexigenic action of ghrelin

Current evidence suggests that ghrelin, a stomach derived peptide, exerts its orexigenic action through specific modulation of Sirtuin1 (SIRT1)/p53 and AMP-activated protein kinase (AMPK) pathways, which ultimately increase the expression of agouti-related protein (AgRP) and neuropeptide Y (NPY) in the arcuate nucleus of the hypothalamus (ARC). However, there is a paucity of data about the possible action of ghrelin on alternative metabolic pathways at this level. Here, we demonstrate that ghrelin elicits a marked upregulation of the hypothalamic mammalian target of rapamycin (mTOR) signaling pathway. Of note, central inhibition of mTOR signaling with rapamycin decreased ghrelin’s orexigenic action and normalized the mRNA expression of AgRP and NPY, as well as their key downstream transcription factors, namely cAMP response-element binding protein (pCREB) and forkhead box O1 (FoxO1, total and phosphorylated). Taken together, these data indicate that, in addition to previous reported mechanisms, ghrelin also promotes feeding through modulation of hypothalamic mTOR pathway.The research leading to these results has received funding from the European Community’s Seventh Framework Programme (FP7/2007–2013) under grant agreement n° 281854 - the ObERStress project (ML) and 245009 - the Neurofast project (RN, CD and ML), Xunta de Galicia (ML: 10PXIB208164PR; RN: 2010/14), Junta de Andalucía (MTS: P08-CVI-03788), Instituto de Salud Carlos III (ISCIII) (ML: PS09/01880), MINECO co-funded by the FEDER Program of EU (MTS: BFU2011-25021; RN: RyC-2008-02219 and SAF2009-07049; ML: RyC-2007-00211; CD: BFU2011-29102). LM is a recipient of a fellowship from Fundação para a Ciência e Tecnologia (FCT), Portugal (SFRH/BD/65379/2009). CIBER de Fisiopatología de la Obesidad y Nutrición is an initiative of ISCIIIS

Sequential Exposure to Obesogenic Factors in Females Rats: From Physiological Changes to Lipid Metabolism in Liver and Mesenteric Adipose Tissue

During their lifetime, females are subjected to different nutritional and hormonal factors that could increase the risk of obesity and associated comorbidities. From early postnatal periods until the postmenopausal phase, exposure to over nutrition, high-energy diet and oestrogen deficiency, are considered as significant obesity risk factors in women. In this study, we assessed how key transitional life events and exposure to different nutrition influence energy homeostasis in a rat model. Specifically, we assessed the sequential exposure to postnatal over nutrition, high-fat diet (HFD) after weaning, followed later by ovariectomy (OVX; as a model of menopause). Each obesity risk factor increased significantly body weight (BW) and adiposity, with additive effects after sequential exposure. Increased energy intake in both HFD and/or OVX groups, and decreased locomotor activity and energy expenditure after OVX can explain these metabolic changes. Our study also documents decreased lipogenic pathway in mesenteric adipose tissue after HFD and/or OVX, independent of previous postnatal programming, yet only HFD evoked this effect in liver. In addition, we report an increase in the expression of the hepatic PEPCK depending on previous metabolic status. Overall, our results identify the impact of different risk factors, which will help in understanding the development of obesity in females

Resistin Regulates Pituitary Lipid Metabolism and Inflammation In Vivo and In Vitro

The adipokine resistin is an insulin-antagonizing factor that also plays a regulatory role in inflammation, immunity, food intake, and gonadal function and also regulates growth hormone (GH) secretion in rat adenopituitary cells cultures with the adipokine. Although adipose tissue is the primary source of resistin, it is also expressed in other tissues, including the pituitary. The aim of this study is to investigate the possible action of resistin on the lipid metabolism in the pituitary gland in vivo (rats in two different nutritional status, fed and fast, treated with resistin on acute and a chronic way) and in vitro (adenopituitary cell cultures treated with the adipokine). Here, by a combination of in vivo and in vitro experimental models, we demonstrated that central acute and chronic administration of resistin enhance mRNA levels of the lipid metabolic enzymes which participated on lipolysis and moreover inhibiting mRNA levels of the lipid metabolic enzymes involved in lipogenesis. Taken together, our results demonstrate for the first time that resistin has a regulatory role on lipid metabolism in the pituitary gland providing a novel insight in relation to the mechanism by which this adipokine can participate in the integrated control of lipid metabolism.Sara Borrell Postdoctoral program; BFU 2011 and CIBER Obesidad y Nutricion (Instituto de Salud Carlos Tercero (ISCIII), Ministerio de Ciencia e Innovacion). Juan de la Cierva Program (Ministerio de Educacion y Ciencia)S

Mad2 inhibits the mitotic kinesin MKlp2

The mitotic checkpoint protein Mad2 halts cell division by interfering with MKlp2-mediated relocation of the chromosome passenger complex from centromeres to the mitotic spindle

Protein Phosphatase 1 inactivates Mps1 to ensure efficient Spindle Assembly Checkpoint silencing

Faithfull genome partitioning during cell division relies on the Spindle Assembly Checkpoint (SAC), a conserved signaling pathway that delays anaphase onset until all chromosomes are attached to spindle microtubules. Mps1 kinase is an upstream SAC regulator that promotes the assembly of an anaphase inhibitor through a sequential multi-target phosphorylation cascade. Thus, the SAC is highly responsive to Mps1, whose activity peaks in early mitosis as a result of its T-loop autophosphorylation. However, the mechanism controlling Mps1 inactivation once kinetochores attach to microtubules and the SAC is satisfied remains unknown. Here we show in vitro and in Drosophila that Protein Phosphatase 1 (PP1) inactivates Mps1 by dephosphorylating its T-loop. PP1-mediated dephosphorylation of Mps1 occurs at kinetochores and in the cytosol, and inactivation of both pools of Mps1 during metaphase is essential to ensure prompt and efficient SAC silencing. Overall, our findings uncover a mechanism of SAC inactivation required for timely mitotic exit.We thank Geert Kops (UMC, Utrecht, The Netherlands), Thomas Maresca (University of Massachu-setts Amherst, USA) and Christian Lehner (University of Zurich, Switzerland) for antibodies, con-structs and fly stocks. We thank Carla Sofia Lopes (i3S, IBMC, University of Porto) and all the members of the Sunkel laboratory for critical and helpful discussions. This article is a result of the project Norte-01-0145-FEDER-000029 - Advancing Cancer Research: From basic knowledge to application, supported by Norte Portugal Regional Operational Programme (NORTE 2020), under the PORTUGAL 2020 Partnership Agreement, through the European Regional Development Fund (FEDER). MO is supported by a fellowship from the GABBA PhD program from the University of Porto, PD/BD/105746/2014. JB is supported by an FCT PhD grant SFRH/BD/87871/2012. CC is sup-ported by an FCT investigator position and funding (IF/01755/2014). HM is funded by PRECISE and CODECHECK grants from the European Research Council, FLAD Life Science 2020, and the Louis-Jeantet Young Investigator Career Award