Activation of G protein-coupled receptors by ketone bodies: Clinical implication of the ketogenic diet in metabolic disorders

Ketogenesis takes place in hepatocyte mitochondria where acetyl-CoA derived from fatty acid catabolism is converted to ketone bodies (KB), namely β-hydroxybutyrate (β-OHB), acetoacetate and acetone. KB represent important alternative energy sources under metabolic stress conditions. Ketogenic diets (KDs) are low-carbohydrate, fat-rich eating strategies which have been widely proposed as valid nutritional interventions in several metabolic disorders due to its substantial efficacy in weight loss achievement. Carbohydrate restriction during KD forces the use of FFA, which are subsequently transformed into KB in hepatocytes to provide energy, leading to a significant increase in ketone levels known as "nutritional ketosis". The recent discovery of KB as ligands of G protein-coupled receptors (GPCR) - cellular transducers implicated in a wide range of body functions - has aroused a great interest in understanding whether some of the clinical effects associated to KD consumption might be mediated by the ketone/GPCR axis. Specifically, anti-inflammatory effects associated to KD regimen are presumably due to GPR109A-mediated inhibition of NLRP3 inflammasome by β-OHB, whilst lipid profile amelioration by KDs could be ascribed to the actions of acetoacetate via GPR43 and of β-OHB via GPR109A on lipolysis. Thus, this review will focus on the effects of KD-induced nutritional ketosis potentially mediated by specific GPCRs in metabolic and endocrinological disorders. To discriminate the effects of ketone bodies per se, independently of weight loss, only studies comparing ketogenic vs isocaloric non-ketogenic diets will be considered as well as short-term tolerability and safety of KDs

La mutazione di PIK3CA nel carcinoma colorettale metastatico: le caratteristiche clinico-patologiche associate e la correlazione con l'outcome.

La prognosi dei pazienti con carcinoma del colon-retto (CRC) è nettamente migliorata negli ultimi anni grazie agli avanzamenti nell'ambito dello screening, della diagnosi e della terapia, oggi sempre più personalizzata. Nel trattamento del tumore del colon-retto, le opzioni di cura oggi disponibili sono molteplici e comprendono trattamenti locoregionali (chirurgia, radioterapia, ablazione) integrati con terapie sistemiche. Il trattamento chirurgico rappresenta la principale opzione terapeutica con intento curativo negli stadi iniziali, in assenza di metastasi a distanza. La chemioterapia adiuvante, in casi selezionati sulla base del rischio, ha dimostrato offrire una riduzione del tasso di mortalità CRC-correlata e della recidiva della malattia rispetto alla sola chirurgia. La valutazione multidisciplinare del singolo paziente è fondamentale per la scelta della strategia ottimale, individualizzata in base a caratteristiche cliniche e tumorali. Allo stesso tempo, lo studio del profilo molecolare del CRC, al fine di individuare fattori prognostici e predittivi, permette un'ulteriore personalizzazione del trattamento. Al di là dei più noti RAS e BRAF, i dati suggeriscono che le mutazioni di PI3K potrebbero ricoprire un importante ruolo in questo ambito. La PI3K (fosfatidilinositolo-3-chinasi) è una chinasi lipidica ubiquitaria, ampiamente espressa nell'organismo, che agisce sia come trasduttore del segnale in differenti vie cellulari a seguito della stimolazione dei recettori presenti sulla superficie cellulare, sia nei processi di smistamento delle proteine. La via di segnalazione di PI3K ha un ruolo importante nelle funzioni cellulari, essendo infatti implicata nel controllo metabolico, nell'immunità, nell'angiogenesi, nell'omeostasi, ma anche nella sopravvivenza, nella proliferazione e nella crescita delle cellule tumorali. L'amplificazione di uno dei geni che codifica per PI3K, PIK3CA, la perdita della fosfatasi PTEN e le mutazioni di AKT, frequentemente associate, promuovono la tumorigenesi attraverso l'up-regolazione della via di segnalazione corrispondente. Le mutazioni del gene PIK3CA, localizzato sul cromosoma 3, coinvolgono prevalentemente negli esoni 9 e 20 e il 15-20% dei tumori del colon-retto. Sulla base di alcune analisi retrospettive, le mutazioni di PIK3CA nel CRC metastatico sembrano essere più frequenti nei tumori del colon prossimale e a carico dell'esone 9. Tali mutazioni, inoltre, sembrerebbero associate ad una minore sopravvivenza libera da recidiva nei pazienti in stadio II/III operati e a una più breve sopravvivenza globale rispetto ai pazienti wild-type, aspetto che conferirebbe a PIK3CA un ruolo prognostico negativo. Le mutazioni del gene PIK3CA sembrano avere anche un ruolo predittivo negativo di risposta ai trattamenti con farmaci anti-EGFR, in particolare quelle localizzate a livello dell'esone 20. L'assunzione regolare dell'aspirina in pazienti con tumore PIK3CA mutato sembra però migliorare la loro prognosi. Attualmente, sono in corso studi con nuovi farmaci che interferiscono con la via di segnalazione di PI3K/AKT/mTOR, come gli inibitori di PI3K, i doppi inibitori di PI3K/mTOR e gli inibitori di AKT, sia in monoterapia, sia in combinazione con farmaci citotossici e a bersaglio molecolare. Complessivamente le informazioni disponibili riguardo le caratteristiche clinico-patologiche associate e il ruolo prognostico e predittivo dei tumori PIK3CA mutati derivano soltanto da studi retrospettivi che hanno riportato risultati contrastanti. Lo scopo di questo studio è cercare, quindi, di fare chiarezza sul ruolo di PI3K nel tumore del colon-retto, dal momento che una conoscenza approfondita della mutazione di PIK3CA permetterebbe di personalizzare ulteriormente l'iter terapeutico. Nella nostra valutazione sono stati inclusi 585 pazienti consecutivi afferiti all'Azienda Ospedaliero-Universitaria Pisana dall'1 gennaio 2005 al 31 dicembre 2017 con diagnosi accertata istologicamente di adenocarcinoma colorettale metastatico, il cui materiale istologico era stato analizzato con MALDI-TOF MassArray come da pratica clinica e di cui erano disponibili informazioni cliniche complete. Di questi, 98 (16.75%) presentavano mutazione di PIK3CA, di cui circa il 60% localizzate sull'esone 9, il 20% sull'esone 20 e il restante 20% su altri esoni (1, 4, 6). Rispetto ai tumori wild-type, i PIK3CA mutati erano più spesso RAS mutati, MSI high e localizzati nel colon di destra. Tra i 53 pazienti dei quali era disponibile lo stato di PI3K sia sul tumore primitivo che sulle metastasi, la concordanza era del 92.4%, in linea con i dati disponibili. Per quanto riguarda il ruolo prognostico della mutazione, non sono state riscontrate differenze significative in termini di overall survival (OS) tra i pazienti PIK3CA mutati e PIK3CA wild-type. In contrasto con altri dati presenti in letteratura, l'uso di aspirina nei pazienti PIK3CA mutati non ha migliorato in modo statisticamente significativo l'OS. Nel sottogruppo di pazienti RAS/BRAF wild-type e RAS mutati, non sono state trovate differenze in termini di OS tra i PIK3CA mutati e i PIK3CA wild-type, nonostante i PIK3CA mutati avessero una OS minore. Anche nel sottogruppo dei pazienti BRAF mutati, la sopravvivenza non variava in modo significativo in base allo stato di PIK3CA. Il ruolo predittivo della mutazione di PIK3CA è stato valutato in relazione all'utilizzo degli anti EGFR, sia in qualsiasi linea e con qualsiasi schedula, sia in prima linea e con qualsiasi schedula, sia in prima linea associati a chemioterapia, sia in monoterapia +/- irinotecano nei pazienti irinotecano-refrattari. In nessun caso sono state dimostrate differenze significative in attività ed efficacia sulla base della mutazione di PIK3CA, ma ciò può essere in parte dovuto alla piccola dimensione del campione. Tale mutazione, perciò, sembra definire un sottoinsieme rilevante di tumori con peculiari caratteristiche cliniche, molecolari e prognostiche. Una analisi più approfondita e su una popolazione più numerosa potrebbe essere utile al fine di rendere più chiari alcuni aspetti di PIK3CA che potrebbero anche determinare un cambiamento per la pratica clinica

In vivo silencing of aquaporin-1 by RNA interference inhibits angiogenesis in the chick embryo chorioallantoic membrane assay

Aquaporin-1(AQP1) is a water channel protein mainly expressed in endothelial and epithelial cells of many tissues, including the vasculature where it serves to increase cell membrane water permeability. Previous studies in active multiple myeloma patients and in AQP1 KO mice indicated an involvement of AQP1 in physiological and tumor angiogenesis. To understand the physiological role of AQP1 in angiogenesis, we used a 21-nucleotide small interfering RNA duplexes (siRNA) to knockdown AQP1 in the chick embryo chorioallantoic membrane (CAM), a commonly used in vivo assay to study both angiogenic and angiostatic molecules. Chicken AQP1 sequence was identified and utilized to synthesize a siRNA directed to the AQP1 sequence. We then tested the efficiency of the siRNA in vitro, using an AQP1 transfected cell line. The level of AQP1 protein reduction obtained using siRNA was 98 % and 92 % after 1 and 2 day transfection respectively. RNA interference experiments were then performed in vivo by using the CAM assay. Results showed that after 4 days of treatment, AQP1 siRNA was able to strongly inhibit angiogenesis. This is the first study showing the in vivo use of RNA interference technique in the CAM assay. Our results strongly support the hypothesis that AQP1 could have a key role in physiological and pathological angiogenesis

The KATP channel is a molecular sensor of atrophy in skeletal muscle

The involvement of ATP-sensitive K+ (K-ATP) channels in the atrophy of slow-twitch (MHC-I) soleus (SOL) and fast-twitch (MHC-IIa) flexor digitorum brevis (FDB) muscles was investigated in vivo in 14-day-hindlimb-unloaded (14-HU) rats, an animal model of disuse, and in vitro in drug-induced muscle atrophy. Patch-clamp and gene expression experiments were performed in combination with measurements of fibre diameters used as an index of atrophy, and with MHC labelling in 14-HU rats and controls. A down-regulation of K-ATP channel subunits Kir6.2, SUR1 and SUR2B with marked atrophy and incomplete phenotype transition were observed in SOL of 14-HU rats. The observed changes in K-ATP currents were well correlated with changes in fibre diameters and SUR1 expression, as well as with MHC-IIa expression. Half of the SOL fibres of 14-HU rats had reduced diameter and K-ATP currents and were labelled by MHC-I antibodies. Non-atrophic fibres were labelled by MHC-IIa (22%) antibodies and had enhanced K-ATP currents, or were labelled by MHC-I (28%) antibodies but had normal current. FDB was not affected in 14-HU rats and this is related to the high expression/activity of Kir6.2/SUR1 subunits characterizing this muscle phenotype. The long-term incubation of the control muscles in vitro with the K-ATP channel blocker glibenclamide (10-6 m) reduced the K-ATP currents with atrophy and these effects were prevented by the K-ATP channel opener diazoxide (10-4 m). The in vivo down-regulation of SUR1, and possibly of Kir6.2 and SUR2B, or their in vitro pharmacological blockade activates atrophic signalling in skeletal muscle. All these findings suggest a new role for the K-ATP channel as a molecular sensor of atrophy.The involvement of ATP-sensitive K+ (KATP) channels in the atrophy of slow-twitch (MHC-I) soleus (SOL) and fast-twitch (MHC-IIa) flexor digitorum brevis (FDB) muscles was investigated in vivo in 14-day-hindlimb-unloaded (14-HU) rats, an animal model of disuse, and in vitro in drug-induced muscle atrophy. Patch-clamp and gene expression experiments were performed in combination with measurements of fibre diameters used as an index of atrophy, and with MHC labelling in 14-HU rats and controls. A down-regulation of KATP channel subunits Kir6.2, SUR1 and SUR2B with marked atrophy and incomplete phenotype transition were observed in SOL of 14-HU rats. The observed changes in KATP currents were well correlated with changes in fibre diameters and SUR1 expression, as well as with MHC-IIa expression. Half of the SOL fibres of 14-HU rats had reduced diameter and KATP currents and were labelled by MHC-I antibodies. Non-atrophic fibres were labelled by MHC-IIa (22%) antibodies and had enhanced KATP currents, or were labelled by MHC-I (28%) antibodies but had normal current. FDB was not affected in 14-HU rats and this is related to the high expression/activity of Kir6.2/SUR1 subunits characterizing this muscle phenotype. The long-term incubation of the control muscles in vitro with the KATP channel blocker glibenclamide (10-6 m) reduced the KATP currents with atrophy and these effects were prevented by the KATP channel opener diazoxide (10-4 m). The in vivo down-regulation of SUR1, and possibly of Kir6.2 and SUR2B, or their in vitro pharmacological blockade activates atrophic signalling in skeletal muscle. All these findings suggest a new role for the KATP channel as a molecular sensor of atrophy. © 2010 The Authors. Journal compilation © 2010 The Physiological Society

Gentamicin treatment in exercised mdx mice: Identification of dystrophin-sensitive pathways and evaluation of efficacy in work-loaded dystrophic muscle

Aminoglycosides force read through of premature stop codon mutations and introduce new mutation-specific gene-corrective strategies in Duchenne muscular dystrophy. A chronic treatment with gentamicin (32 mg/kg/daily i.p., 8-12 weeks) was performed in exercised mdx mice with the dual aim to clarify the dependence on dystrophin of the functional, biochemical and histological alterations present in dystrophic muscle and to verify the long term efficiency of small molecule gene-corrective strategies in work-loaded dystrophic muscle. The treatment counteracted the exercise-induced impairment of in vivo forelimb strength after 6-8 weeks. We observed an increase in dystrophin expression level in all the fibers, although lower than that observed in normal fibers, and found a concomitant recovery of aquaporin-4 at sarcolemma. A significant reduction in centronucleated fibers, in the area of necrosis and in the percentage of nuclear factor-kB-positive nuclei was observed in gastrocnemious muscle of treated animals. Plasma creatine kinase was reduced by 70%. Ex vivo, gentamicin restored membrane ionic conductance in mdx diaphragm and limb muscle fibers. No effects were observed on the altered calcium homeostasis and sarcolemmal calcium permeability, detected by electrophysiological and microspectrofluorimetric approaches. Thus, the maintenance of a partial level of dystrophin is sufficient to reinforce sarcolemmal stability, reducing leakiness, inflammation and fiber damage, while correction of altered calcium homeostasis needs greater expression of dystrophin or direct interventions on the channels involved. © 2008 Elsevier Inc. All rights reserved

p66Shc regulates vesicle-mediated secretion in mast cells by affecting F-actin dynamics

p66Shc is recruited as a complex with SHIP-1 to the F-actin skeleton, and negatively regulates mast cell secretion and membrane ruffling by impairing F-actin dynamics. The extracellular vesicular compartment has emerged as a novel system of intercellular communication; however, the mechanisms involved in membrane vesicle biogenesis and secretion are as yet unclear. Among immune cells releasing membrane vesiclesmast cells that reside near tissues exposed to the environmentare master modulators of immune responses. Here, we have addressed the role of p66Shc, a novel regulator of mast cell activation and homeostasis, in the dynamic reorganization of the actin cytoskeleton that is associated with morphological changes during secretion. We show that p66Shc is recruited as a complex with the lipid phosphatase SHIP1 to the F-actin skeleton and impairs antigen-dependent cortical F-actin disassembly and membrane ruffling through the inhibition of Vav and paxillin phosphorylation. We also show that in addition to acting as a negative regulator of antigen-dependent mast cell degranulation, p66Shc limits the basal release of granule contents by inhibiting microvesicle budding from the plasma membrane and piecemeal degranulation. These findings identify p66Shc as a critical regulator of actin dynamics in mast cells, providing a basis for understanding the molecular mechanisms involved in vesicle-mediated secretion in these cells

Real-World Outcome of Treatment with Single-Agent Ibrutinib in Italian Patients with Chronic Lymphocytic Leukemia: Final Results of the EVIdeNCE Study

Simple Summary In clinical trials, ibrutinib was found to be effective and well-tolerated in patients with chronic lymphocytic leukemia (CLL). To confirm these findings, data on unselected patients treated in clinical practice are necessary. The aim of our observational, prospective Italian cohort study was to describe the real-world persistence rate, patterns of use, and clinical outcomes in patients with CLL treated with single-agent ibrutinib across various treatment lines. We found that, despite the high burden of patient comorbidities and unfavorable genetic features, the majority of patients (217/309, 70%), especially those treated in first line (75%), continued ibrutinib treatment for >= 2 years. The most common reasons for treatment discontinuation were adverse events, primarily infections. We reported positive clinical and survival outcomes, especially in the first-line cohort, and a safety profile consistent with clinical trial data. Our data suggest that ibrutinib is a valuable option for both treatment-naive and previously treated patients with CLL.Abstract Real-world data in clinical practice are needed to confirm the efficacy and safety that ibrutinib has demonstrated in clinical trials of patients with chronic lymphocytic leukemia (CLL). We described the real-world persistence rate, patterns of use, and clinical outcomes in 309 patients with CLL receiving single-agent ibrutinib in first line (1L, n = 118), 2L (n = 127) and >= 3L (n = 64) in the prospective, real-world, Italian EVIdeNCE study. After a median follow-up of 23.9 months, 29.8% of patients discontinued ibrutinib (1L: 24.6%, 2L: 29.9%, >= 3L: 39.1%), mainly owing to adverse events (AEs)/toxicity (14.2%). The most common AEs leading to discontinuation were infections (1L, >= 3L) and cardiac events (2L). The 2-year retention rate was 70.2% in the whole cohort (1L: 75.4%, 2L: 70.1%, >= 3L: 60.9%). The 2-year PFS and OS were, respectively, 85.4% and 91.7% in 1L, 80.0% and 86.2% in 2L, and 70.1% and 80.0% in >= 3L. Cardiovascular conditions did not impact patients' clinical outcomes. The most common AEs were infections (30.7%), bleeding (12.9%), fatigue (10.0%), and neutropenia (9.7%), while grade 3-4 atrial fibrillation occurred in 3.9% of patients. No new safety signals were detected. These results strongly support ibrutinib as a valuable treatment option for CLL