Food perception promotes phosphorylation of MFFS131 and mitochondrial fragmentation in liver.

Liver mitochondria play a central role in metabolic adaptations to changing nutritional states, yet their dynamic regulation upon anticipated changes in nutrient availability has remained unaddressed. Here, we found that sensory food perception rapidly induced mitochondrial fragmentation in the liver through protein kinase B/AKT (AKT)-dependent phosphorylation of serine 131 of the mitochondrial fission factor (MFFS131). This response was mediated by activation of hypothalamic pro-opiomelanocortin (POMC)-expressing neurons. A nonphosphorylatable MFFS131G knock-in mutation abrogated AKT-induced mitochondrial fragmentation in vitro. In vivo, MFFS131G knock-in mice displayed altered liver mitochondrial dynamics and impaired insulin-stimulated suppression of hepatic glucose production. Thus, rapid activation of a hypothalamus-liver axis can adapt mitochondrial function to anticipated changes of nutritional state in control of hepatic glucose metabolism.

Insulin signalling in tanycytes gates hypothalamic insulin uptake and regulation of AgRP neuron activity.

Insulin acts on neurons and glial cells to regulate systemic glucose metabolism and feeding. However, the mechanisms of insulin access in discrete brain regions are incompletely defined. Here we show that insulin receptors in tanycytes, but not in brain endothelial cells, are required to regulate insulin access to the hypothalamic arcuate nucleus. Mice lacking insulin receptors in tanycytes (IR∆Tan mice) exhibit systemic insulin resistance, while displaying normal food intake and energy expenditure. Tanycytic insulin receptors are also necessary for the orexigenic effects of ghrelin, but not for the anorexic effects of leptin. IR∆Tan mice exhibit increased agouti-related peptide (AgRP) neuronal activity, while displaying blunted AgRP neuronal adaptations to feeding-related stimuli. Lastly, a highly palatable food decreases tanycytic and arcuate nucleus insulin signalling to levels comparable to those seen in IR∆Tan mice. These changes are rooted in modifications of cellular stress responses and of mitochondrial protein quality control in tanycytes. Conclusively, we reveal a critical role of tanycyte insulin receptors in gating feeding-state-dependent regulation of AgRP neurons and systemic insulin sensitivity, and show that insulin resistance in tanycytes contributes to the pleiotropic manifestations of obesity-associated insulin resistance.

Orexin receptors 1 and 2 in serotonergic neurons differentially regulate peripheral glucose metabolism in obesity.

The wake-active orexin system plays a central role in the dynamic regulation of glucose homeostasis. Here we show orexin receptor type 1 and 2 are predominantly expressed in dorsal raphe nucleus-dorsal and -ventral, respectively. Serotonergic neurons in ventral median raphe nucleus and raphe pallidus selectively express orexin receptor type 1. Inactivation of orexin receptor type 1 in serotonin transporter-expressing cells of mice reduced insulin sensitivity in diet-induced obesity, mainly by decreasing glucose utilization in brown adipose tissue and skeletal muscle. Selective inactivation of orexin receptor type 2 improved glucose tolerance and insulin sensitivity in obese mice, mainly through a decrease in hepatic gluconeogenesis. Optogenetic activation of orexin neurons in lateral hypothalamus or orexinergic fibers innervating raphe pallidus impaired or improved glucose tolerance, respectively. Collectively, the present study assigns orexin signaling in serotonergic neurons critical, yet differential orexin receptor type 1- and 2-dependent functions in the regulation of systemic glucose homeostasis.

Mild Impairment of Mitochondrial OXPHOS Promotes Fatty Acid Utilization in POMC Neurons and Improves Glucose Homeostasis in Obesity.

Mitochondrial oxidative phosphorylation (OXPHOS) and substrate utilization critically regulate the function of hypothalamic proopiomelanocortin (POMC)-expressing neurons. Here, we demonstrate that inactivation of apoptosis-inducing factor (AIF) in POMC neurons mildly impairs mitochondrial respiration and decreases firing of POMC neurons in lean mice. In contrast, under diet-induced obese conditions, POMC-Cre-specific inactivation of AIF prevents obesity-induced silencing of POMC neurons, translating into improved glucose metabolism, improved leptin, and insulin sensitivity, as well as increased energy expenditure in AIFΔPOMC mice. On a cellular level, AIF deficiency improves mitochondrial morphology, facilitates the utilization of fatty acids for mitochondrial respiration, and increases reactive oxygen species (ROS) formation in POMC neurons from obese mice, ultimately leading to restored POMC firing upon HFD feeding. Collectively, partial impairment of mitochondrial function shifts substrate utilization of POMC neurons from glucose to fatty acid metabolism and restores their firing properties, resulting in improved systemic glucose and energy metabolism in obesity.

Methylation of L1RE1, RARB, and RASSF1 function as possible biomarkers for the differential diagnosis of lung cancer.

BACKGROUND: Lung cancer is the major cause of cancer-related deaths worldwide. Differential diagnosis can be difficult, especially when only small samples are available. Epigenetic changes are frequently tissue-specific events in carcinogenesis and hence may serve as diagnostic biomarkers. MATERIAL AND METHODS: 138 representative formalin-fixed, paraffin-embedded (FFPE) tissues (116 lung cancer cases and 22 benign controls) were used for targeted DNA methylation analysis via pyrosequencing of ten literature-derived methylation markers (APC, CDH1, CDKN2A, EFEMP1, FHIT, L1RE1, MGMT, PTEN, RARB, and RASSF1). Methylation levels were analyzed with the Classification and Regression Tree Algorithm (CART), Conditional Interference Trees (ctree) and ROC. Validation was performed with additional 27 lung cancer cases and 38 benign controls. TCGA data for 282 lung cancer cases was included in the analysis. RESULTS: CART and ctree analysis identified the combination of L1RE1 and RARB as well as L1RE1 and RASSF1 as independent methylation markers with high discriminative power between tumor and benign tissue (for each combination, 91% specificity and 100% sensitivity). L1RE1 methylation associated significantly with tumor type and grade (p<0.001) with highest methylation in the control group. The opposite was found for RARB (p<0.001). RASSF1 methylation increased with tumor type and grade (p<0.001) with strongest methylation in neuroendocrine tumors (NET). CONCLUSION: Hypomethylation of L1RE1 is frequent in tumors compared to benign controls and associates with higher grade, whereas increasing methylation of RARB is an independent marker for tumors and higher grade. RASSF1 hypermethylation was frequent in tumors and most prominent in NET making it an auxiliary marker for separation of NSCLC and NET. L1RE1 in combination with either RARB or RASSF1 could function as biomarkers for separating lung cancer and non-cancerous tissue and could be useful for samples of limited size such as biopsies.

Reviewing the current evidence supporting early B-cells as the cellular origin of Merkel cell carcinoma.

Merkel cell carcinoma (MCC) is a highly malignant skin cancer characterized by early metastases and poor survival. Although MCC is a rare malignancy, its incidence is rapidly increasing in the U.S. and Europe. The discovery of the Merkel cell polyomavirus (MCPyV) has enormously impacted our understanding of its etiopathogenesis and biology. MCCs are characterized by trilinear differentiation, comprising the expression of neuroendocrine, epithelial and B-lymphoid lineage markers. To date, it is generally accepted that the initial assumption of MCC originating from Merkel cells (MCs) is unlikely. This is owed to their post-mitotic character, absence of MCPyV in MCs and discrepant protein expression pattern in comparison to MCC. Evidence from mouse models suggests that epidermal/dermal stem cells might be of cellular origin in MCC. The recently formulated hypothesis of MCC originating from early B-cells is based on morphology, the consistent expression of early B-cell lineage markers and the finding of clonal immunoglobulin chain rearrangement in MCC cells. In this review we elaborate on the cellular ancestry of MCC, the identification of which could pave the way for novel and more effective therapeutic regimens.

[Merkel cell carcinoma: cutaneous manifestation of a highly malignant pre-/pro-B cell neoplasia? : Novel concept about the cellular origin of Merkel cell carcinoma]. / Merkelzellkarzinom: kutane Manifestation einer hochmalignen Prä-/pro-B-Zell-Neoplasie? : Neues Konzept zum zellulären Ursprung des Merkelzellkarzinoms.

Merkel cell carcinoma (MCC) is a relatively rare but highly malignant non-melanoma skin cancer of the elderly and immunosuppressed patients. The discovery of the Merkel cell polyomavirus (MCPyV) in 2008 significantly impacted the understanding of the etiopathogenesis of MCC. MCPyV is clonally integrated into the MCC genome and approximately 80% of MCC are MCPyV-positive. Recent results of clinical trials using blockade of the PD-1 immune modulatory pathway are promising for the future treatment of MCC. Despite this major progress of the past few years, the cellular origin of MCC still remains obscure. Based on histomorphology, gene expression profiling, and molecular analyses, we have recently hypothesized that MCC originates from pre­/pro-B cells. Here we review putative cells of MCC, including Merkel cells, (epi­)dermal stem cells, and pro­/pre-B cells. In the present work, the focus is on the concept of pre­/pro-B cells as the cellular origin of MCC, which might also impact the understanding of other human small cell malignancies of unknown cellular origin, such as small cell carcinomas of the lung and other anatomical locations. In addition, this concept might pave the way for novel treatment options, especially for advanced MCC.

Preliminary stop of the TOPical Imiquimod treatment of high-grade Cervical intraepithelial neoplasia (TOPIC) trial.

The "TOPical Imiquimod treatment of high-grade Cervical intraepithelial neoplasia" (TOPIC) trial was stopped preliminary, due to lagging inclusions. This study aimed to evaluate the treatment efficacy and clinical applicability of imiquimod 5% cream in high-grade cervical intraepithelial neoplasia (CIN). The lagging inclusions were mainly due to a strong patient preference for either of the two treatment modalities. This prompted us to initiate a new study on the same subject, with a non-randomized, open-label design: the 'TOPical Imiquimod treatment of high-grade Cervical intraepithelial neoplasia (TOPIC)-3' study. Original TOPIC-trial: Medical Ethics Committee approval number METC13231; ClinicalTrials.gov Identifier: NCT02329171, 22 December 2014. TOPIC-3 study: Medical Ethics Committee approval number METC162025; ClinicalTrials.gov Identifier: NCT02917746, 16 September 2016.

Insulin-Dependent Activation of MCH Neurons Impairs Locomotor Activity and Insulin Sensitivity in Obesity.

Melanin-concentrating-hormone (MCH)-expressing neurons (MCH neurons) in the lateral hypothalamus (LH) are critical regulators of energy and glucose homeostasis. Here, we demonstrate that insulin increases the excitability of these neurons in control mice. In vivo, insulin promotes phosphatidylinositol 3-kinase (PI3K) signaling in MCH neurons, and cell-type-specific deletion of the insulin receptor (IR) abrogates this response. While lean mice lacking the IR in MCH neurons (IRΔMCH) exhibit no detectable metabolic phenotype under normal diet feeding, they present with improved locomotor activity and insulin sensitivity under high-fat-diet-fed, obese conditions. Similarly, obesity promotes PI3 kinase signaling in these neurons, and this response is abrogated in IRΔMCH mice. In turn, acute chemogenetic activation of MCH neurons impairs locomotor activity but not insulin sensitivity. Collectively, our experiments reveal an insulin-dependent activation of MCH neurons in obesity, which contributes via distinct mechanisms to the manifestation of impaired locomotor activity and insulin resistance.

TOPical Imiquimod treatment of high-grade Cervical intraepithelial neoplasia (TOPIC trial): study protocol for a randomized controlled trial.

BACKGROUND: Cervical intraepithelial neoplasia (CIN) is the premalignant condition of cervical cancer. Whereas not all high grade CIN lesions progress to cervical cancer, the natural history and risk of progression of individual lesions remain unpredictable. Therefore, high-grade CIN is currently treated by surgical excision: large loop excision of the transformation zone (LLETZ). This procedure has potential complications, such as acute haemorrhage, prolonged bleeding, infection and preterm birth in subsequent pregnancies. These complications could be prevented by development of a non-invasive treatment modality, such as topical imiquimod treatment. The primary study objective is to investigate the efficacy of topical imiquimod 5% cream for the treatment of high-grade CIN and to develop a biomarker profile to predict clinical response to imiquimod treatment. Secondary study objectives are to assess treatment side-effects, disease recurrence and quality of life during and after different treatment modalities. METHODS/DESIGN: The study design is a randomized controlled trial. One hundred forty women with a histological diagnosis of high-grade CIN (CIN 2-3) will be randomized into two arms: imiquimod treatment during 16 weeks (experimental arm) or immediate LLETZ (standard care arm). Treatment efficacy will be evaluated by colposcopy with diagnostic biopsies at 20 weeks for the experimental arm. Successful imiquimod treatment is defined as regression to CIN 1 or less, successful LLETZ treatment is defined as PAP 1 after 6 months. Disease recurrence will be evaluated by cytology at 6, 12 and 24 months after treatment. Side-effects will be evaluated using a standardized report form. Quality of life will be evaluated using validated questionnaires at baseline, 20 weeks and 1 year after treatment. Biomarkers, reflecting both host and viral factors in the pathophysiology of CIN, will be tested at baseline with the aim of developing a predictive biomarker profile for the clinical response to imiquimod treatment. DISCUSSION: Treatment of high-grade CIN lesions with imiquimod in a selected patient population may diminish complications as a result of surgical intervention. More knowledge on treatment efficacy, side effects and long-term recurrence rates after treatment is necessary. TRIAL REGISTRATION: EU Clinical Trials Register EU-CTR2013-001260-34 . Registered 18 March 2013. Medical Ethical Committee approval number: NL44336.068.13 (Medical Ethical Committee Maastricht University Hospital, University of Maastricht). Affiliation: Maastricht University Hospital. Registration number ClinicalTrials.gov: NCT02329171.

Downregulation of MTSS1 expression is an independent prognosticator in squamous cell carcinoma of the lung.

BACKGROUND: The metastasis suppressor 1 (MTSS1) is a newly discovered protein putatively involved in tumour progression and metastasis. MATERIAL AND METHODS: Immunohistochemical expression of MTSS1 was analysed in 264 non-small-cell lung carcinomas (NSCLCs). RESULTS: The metastasis suppressor 1 was significantly overexpressed in NSCLC compared with normal lung (P=0.01). Within NSCLC, MTSS1 expression was inversely correlated with pT-stage (P=0.019) and histological grading (P<0.001). NSCLC with MTSS1 downregulation (<20%) showed a significantly worse outcome (P=0.007). This proved to be an independent prognostic factor in squamous cell carcinomas (SCCs; P=0.041), especially in early cancer stages (P=0.006). CONCLUSION: The metastasis suppressor 1 downregulation could thus serve as a stratifying marker for adjuvant therapy in early-stage SCC of the lung.

[The importance of an early accompanying evaluation of new care forms for the development of indicators for quality assurance in outpatient psychiatric integrated care]. / Die Bedeutung einer frühzeitigen begleitenden Evaluation für die Ableitung von Indikatoren zur Qualitätsmessung in der ambulanten psychiatrischen Integrierten Versorgung.

Aim of this contribution is to illustrate the imp-ortance of an early accompanying evaluation of new care forms for the development of indicators. The illustration uses the experience of the accompanying evaluation of the integrated care model for optimisation of outpatient psychiatric care. For the integrated care model we could develop potential indicators by using medical-psychiatric and insured-related routine data, but all potential indicators need further development to enable reliable statements about achieved quality targets. It is shown that the development of indicators in the outpatient psychiatric integrated care is affected by many different factors such as vague target agreements in the contract and missing contractual agreements for the data. As a result it is illustrated that in this project the evaluation was introduced after implementation of this new form of care and the already established contract and the data management impeded the development of indicators.

Signaling by IL-6 promotes alternative activation of macrophages to limit endotoxemia and obesity-associated resistance to insulin.

Obesity and resistance to insulin are closely associated with the development of low-grade inflammation. Interleukin 6 (IL-6) is linked to obesity-associated inflammation; however, its role in this context remains controversial. Here we found that mice with an inactivated gene encoding the IL-6Rα chain of the receptor for IL-6 in myeloid cells (Il6ra(Δmyel) mice) developed exaggerated deterioration of glucose homeostasis during diet-induced obesity, due to enhanced resistance to insulin. Tissues targeted by insulin showed increased inflammation and a shift in macrophage polarization. IL-6 induced expression of the receptor for IL-4 and augmented the response to IL-4 in macrophages in a cell-autonomous manner. Il6ra(Δmyel) mice were resistant to IL-4-mediated alternative polarization of macrophages and exhibited enhanced susceptibility to lipopolysaccharide (LPS)-induced endotoxemia. Our results identify signaling via IL-6 as an important determinant of the alternative activation of macrophages and assign an unexpected homeostatic role to IL-6 in limiting inflammation.

Neonatal insulin action impairs hypothalamic neurocircuit formation in response to maternal high-fat feeding.

Maternal metabolic homeostasis exerts long-term effects on the offspring's health outcomes. Here, we demonstrate that maternal high-fat diet (HFD) feeding during lactation predisposes the offspring for obesity and impaired glucose homeostasis in mice, which is associated with an impairment of the hypothalamic melanocortin circuitry. Whereas the number and neuropeptide expression of anorexigenic proopiomelanocortin (POMC) and orexigenic agouti-related peptide (AgRP) neurons, electrophysiological properties of POMC neurons, and posttranslational processing of POMC remain unaffected in response to maternal HFD feeding during lactation, the formation of POMC and AgRP projections to hypothalamic target sites is severely impaired. Abrogating insulin action in POMC neurons of the offspring prevents altered POMC projections to the preautonomic paraventricular nucleus of the hypothalamus (PVH), pancreatic parasympathetic innervation, and impaired glucose-stimulated insulin secretion in response to maternal overnutrition. These experiments reveal a critical timing, when altered maternal metabolism disrupts metabolic homeostasis in the offspring via impairing neuronal projections, and show that abnormal insulin signaling contributes to this effect.

K(ATP)-channel-dependent regulation of catecholaminergic neurons controls BAT sympathetic nerve activity and energy homeostasis.

Brown adipose tissue (BAT) is a critical regulator of glucose, lipid, and energy homeostasis, and its activity is tightly controlled by the sympathetic nervous system. However, the mechanisms underlying CNS-dependent control of BAT sympathetic nerve activity (SNA) are only partly understood. Here, we demonstrate that catecholaminergic neurons in the locus coeruleus (LC) adapt their firing frequency to extracellular glucose concentrations in a K(ATP)-channel-dependent manner. Inhibiting K(ATP)-channel-dependent control of neuronal activity via the expression of a variant K(ATP) channel in tyrosine-hydroxylase-expressing neurons and in neurons of the LC enhances diet-induced obesity in mice. Obesity results from decreased energy expenditure, lower steady-state BAT SNA, and an attenuated ability of centrally applied glucose to activate BAT SNA. This impairs the thermogenic transcriptional program of BAT. Collectively, our data reveal a role of K(ATP)-channel-dependent neuronal excitability in catecholaminergic neurons in maintaining thermogenic BAT sympathetic tone and energy homeostasis.

Pitfalls and technical aspects during the research of intestinal anastomotic healing in rats.

BACKGROUND: Fundamental experimental research into intestinal anastomotic healing in rodent models will gain increasing interest in the future. METHODS: The aim of this study was to describe our 5-year experience with a standardized experimental setup of small and large bowel anastomoses in a rodent model and present a basic set of assessment tools investigating anastomotic healing. Anastomotic technique, perioperative complications such as anastomotic insufficiency (AI) and obstructive ileus were in the focus. RESULTS: During different studies with varying study patterns, 167 rat small bowel anastomoses and 120 colonic anastomoses were performed. Overall mortality was 3.6% in small bowel and 2.5% in colonic anastomoses, AI occurred in 2.9 and 4%, respectively. A postoperative obstructive ileus was seen in 3/167 small bowel anastomoses and none in the colonic group. CONCLUSION: When performing experimental intestinal anastomoses in a standardized operative setting and critically considering special perioperative issues, the incidence of relevant complications can be maintained at an adequately low level.

[Merkel cell polyomavirus in the pathogenesis of non-melanoma skin cancer]. / Das Merkel-Zell-Polyomavirus in der Pathogenese nichtmelanozytärer Hauttumoren.

Merkel cell carcinoma (MCC) is a very aggressive neuroendocrine carcinoma of the skin. The recently identified Merkel cell polyomavirus (MCPyV) is present in the majority of MCCs. MCPyV clonally integrates in the tumor DNA and tumor-specific viral mutations are detected within the large T-antigen. To elucidate a possible role of MCPyV in the pathogenesis of other non-melanoma skin cancers (NMSC), i.e. squamous cell carcinoma, Bowen's disease and basal cell carcinoma we tested a group of these tumors in immunosuppressed and immunocompetent patients. In addition we tested MCPyV-positive tumors for viral mutations within the large T-antigen. MCPyV DNA was significantly more frequently detected in the NMSC of the immunosuppressed patients (p<0.001). No tumor specific mutations were found within the large T-antigen. The presence of the virus in tumor cells was confirmed by FISH analysis. Although MCPyV is present in the tumor cells of squamous cell carcinoma, Bowen's disease and basal cell skin carcinoma, further investigations into the role of MCPyV in the pathogenesis of these tumors is needed.

Performance of a polymer-based DNA chip platform in detection and genotyping of human papillomavirus in clinical samples.

Human papillomavirus (HPV) plays a key role in the development of cervical and laryngeal cancers. The aim of our study was to compare the performance of a new hydrogel-based HPV genotyping biochip assay (Biochip) to a commercially available and CE-marked conventional PCR followed by reverse hybridization (GenID-PCR). One hundred twenty-three samples were available for the study. Of these samples, 101/123 were gynecological swabs, 8/123 were swabs or biopsy samples of genital warts, 7/123 were biopsy samples of otorhinolaryngeal lesions, 5/123 were samples of skin warts, and 2/123 were samples of orolabial abnormalities. These molecular methods for HPV genotyping showed comparable sensitivity and specificity. However, 19/123 of the results were discrepant. Specifically, Biochip showed better performance in the detection of multiple infections, especially when more than one high-risk genotype was present. Due to the different probe configurations used in the two assays, GenID-PCR achieves only group-specific detection of many HPV genotypes, whereas Biochip allows for specific identification. Overall, the newly developed HPV chip system (Biochip) proved to be a suitable tool for HPV detection and genotyping; it also proved to be superior for establishing HPV genotyping methods.

'Light-chain escape-multiple myeloma'-an escape phenomenon from plateau phase: report of the largest patient series using LC-monitoring.

PURPOSE: More intensive and novel therapy options in multiple myeloma (MM) hold the promise to improve treatment outcome. However, disease evolution, induced with long disease duration and extensive pretreatment, has resulted in changes in the biological behaviour of MM and unusual relapse emergence, such as of extramedullary (EM) disease or a shift in secretion from intact immunoglobulin (Ig) to free-light chains (FLCs) only. METHODS: We studied ten patients since 2004, thoroughly assessed relevant patient characteristics, prominent similarities, SFLC-changes, therapy response, mode and speed of progression, and the incidence of light-chain escape (LCE)-MM within our entire myeloma patient cohort. Serum FLCs (SFLCs) were determined via Freelite-assay (Dade-Behringer Nephelometer). RESULTS: This report summarizes the to date largest series of ten patients, whose MM appeared stable, as judged by conventional monitoring of intact Ig levels, but developed severe organ dysfunction as a consequence of initially undetected LC-progression. Median number of anti-MM cycles before LCE occurrence was six, including autologous and/or allogeneic stem cell transplants and novel drugs, predominantly thalidomide, in 4/10. Classic diagnostics, such as electrophoresis and quantitative Ig measurement proved futile to detect LC-progression, whereas SFLCs were reliable markers. The LCE-MM prevalence within 407 MM patients treated in our institution between 2004 and 2007 was 2.46%. CONCLUSIONS: Our report suggests that early detection of LCE-MM by means of serial SFLC measurements may prevent unnecessary complications, allows to detect unusual relapse manifestations in the era of intensive and biological therapy options and possibly also permits to improve treatment results in LCE-MM.