Manipulating mitochondrial dynamics in the NTS prevents diet-induced deficits in brown fat morphology and glucose uptake.

AIMS: Brown adipose tissue (BAT) can produce heat by metabolizing glucose and fatty acids. Activation of BAT is controlled by the central nervous system (CNS) through sympathetic innervation. Dysregulation of signalling molecules in selective CNS areas such as the nucleus of tractus solitarius (NTS) are linked with altered BAT activity, obesity and diabetes. High-fat diet (HFD)-feeding increases mitochondrial fragmentation in the NTS, triggering insulin resistance, hyperphagia and weight gain. Here we sought to determine whether changes in mitochondrial dynamics in the NTS can affect BAT glucose uptake. MAIN METHODS: Rats received DVC stereotactic surgery for local brain administration of viruses that express mutated Drp1 genes. BAT glucose uptake was measured with PET/CT scans. Biochemical assays and immunohistochemistry determined altered levels of key signalling molecules and neural innervation of BAT. KEY FINDINGS: We show that short-term HFD-feeding decreases BAT glucose uptake. However, inhibiting mitochondrial fragmentation in NTS-astrocytes of HFD-fed rats partially restores BAT glucose uptake accompanied by lower blood glucose and insulin levels. Tyrosine Hydroxylase (TH) revealed that rats with inhibited mitochondrial fragmentation in NTS astrocytes had higher levels of catecholaminergic innervation in BAT compared to HFD-fed rats, and did not exhibit HFD-dependent infiltration of enlarged white fat droplets in the BAT. In regular chow-fed rats, increasing mitochondrial fragmentation in the NTS-astrocytes reduced BAT glucose uptake, TH immune-positive boutons and ß3-adrenergic receptor levels. SIGNIFICANCE: Our data suggest that targeting mitochondrial dynamics in the NTS-astrocytes could be a beneficial strategy to increase glucose utilization and protect from developing obesity and diabetes.

Infection-induced extracellular vesicles evoke neuronal transcriptional and epigenetic changes.

Infection with the protozoan Toxoplasma gondii induces changes in neurotransmission, neuroinflammation, and behavior, yet it remains elusive how these changes come about. In this study we investigated how norepinephrine levels are altered by infection. TINEV (Toxoplasma-induced neuronal extracellular vesicles) isolated from infected noradrenergic cells down-regulated dopamine ß-hydroxylase (DBH) gene expression in human and rodent cells. Here we report that intracerebral injection of TINEVs into the brain is sufficient to induce DBH down-regulation and distrupt catecholaminergic signalling. Further, TINEV treatment induced hypermethylation upstream of the DBH gene. An antisense lncRNA to DBH was found in purified TINEV preparations. Paracrine signalling to induce transcriptional gene silencing and DNA methylation may be a common mode to regulate neurologic function.

Digital real-time microscopy of ex-vivo tissues: A novel strategy to control surgical accuracy.

INTRODUCTION: Ex-vivo FCM is a novel digital optical technique that provides images of fresh tissues in a real-time fashion with magnification to subcellular details of a flattened unprocessed sample. Digital images are hematoxylin-eosin-like and can be shared and interpreted remotely. In urology, FCM has been successfully applied for prostate tissue interpretation, either during biopsy and radical prostatectomy. Possible applications of FCM may reflect those of frozen section analysis and can be extended to all fields in which the intra-operative microscopical control is advisable. MATERIALS AND METHODS: This is an investigative prospective case series that aims to explore FCM feasibility in novel surgical settings and provide a depiction of FCM digital images in those fields. The definite purpose is to check the accuracy of surgical specimen during the following interventions: (a) trans-urethral resection of bladder tumors, to confirm the presence of muscular layer; (b) biopsy of a retroperitoneal mass, to check for the location and quality of cores; (c) training in robotic radical prostatectomy, to control surgical margins after a nerve sparing performed by a trainee. To this aim, we collected FCM images during seven surgical procedures. FCM findings were compared to those from the final histopathological analysis and the agreement was assessed. RESULTS: In all cases, FCM digital images were obtained in the OR. FCM was able to confirm the presence of muscular layer in TURB specimen, the presence of lymphomatous tissue, surgical margins at prostate specimen. FCM intra-operative interpretation was consistent with final histopathology in all cases. CONCLUSIONS: Ex vivo FCM may represent a novel approach to control the quality of specimens, likely to tailor surgical strategy in a real-time fashion. Moreover, digitalization represents a step toward the implementation of telepathology in clinical practice.

Peripheral Blood Lymphocyte Percentage May Predict Chemotolerance and Survival in Patients with Advanced Pancreatic Cancer. Association between Adaptive Immunity and Nutritional State.

Pancreatic Carcinoma (PC) cells have the ability to induce patient immunosuppression and to escape immunosurveillance. Low circulating lymphocytes are associated with an advanced stage of PC and reduced survival. Blood lymphocytes expressed as a percentage of Total White Blood Cells (L% TWBC) could predict chemotolerance (n° of tolerated cycles), survival time and Body Weight (BW) more effectively than lymphocytes expressed as an absolute value (LAB > 1500 n°/mm3) or lymphocytes >22%, which is the lowest limit of normal values in our laboratory. Forty-one patients with advanced PC, treated with chemotherapy, were selected for this observational retrospective study. Patients were evaluated at baseline (pre-chemotherapy), and at 6, 12 and 18 months, respectively, after diagnosis of PC. The study found L ≥ 29.7% to be a better predictor of survival (COX model, using age, sex, BW, serum creatinine, bilirubin and lymphocytes as covariates), chemotolerance (r = +0.50, p = 0.001) and BW (r = +0.35, p = 0.027) than LAB > 1500 or L > 22%. BW did not significantly correlate with chemotolerance or survival. The preliminary results of this study suggest that L ≥ 29.7% is more effective than LAB > 1500 or L > 22% at predicting chemotolerance, survival time and nutritional status. A possible impact of nutritional status on chemotherapy and survival seems to be lymphocyte-mediated given the association between BW and L%. This study may serve as the basis for future research to explore whether nutritional interventions can improve lymphopenia, and if so, how this may be possible.

COVID-19 and slowdown of residents' activity: Feedback from a novel e-learning event and overview of the literature.

OBJECTIVE: To evaluate the impact of an e-learning online event, created for supporting resident's training during the slowdown of surgical and clinical activities caused by COVID-19 pandemic. An overview of PubMed literature depicting the state of the art of urology residency in the COVID-19 era was performed as well, to contextualize the issue. METHODS: An online learning event for residents was set up at the beginning of the pandemic; the faculty consisted of experts in urology who provided on-line lectures and videos on surgical anatomy, procedures, updates in guidelines, technology, training. The audience was composed of 30-500 attendees from Italy, USA, India and Belgium. A questionnaire to analyze relevance, satisfaction and popularity of the lessons was mailed to 30 local residents. RESULTS AND LIMITATIONS: Almost all residents defined the web environment suitable to achieve the learning outcomes; the method, the number and the competence of the faculty were appropriate/excellent. Most of the younger residents (81.8%) stated their surgical knowledge would improve after the course; 72.7% declared they would take advantage into routine inpatients clinical activity. Nineteen more expert residents agreed that the course would improve their surgical knowledge and enhance their practical skills; almost all stated that the initiative would change their outpatients and inpatients practice. Overall, 44 articles available in PubMed have addressed the concern of urological learning and training during the pandemic from different standpoints; four of them considered residents' general perception towards web-based learning programs. CONCLUSIONS: The paper confirms residents' satisfaction with e-learning methods and, to our knowledge, is the first one focusing on a specific event promptly settled up at the beginning of the outbreak. Web-based educational experience developed during the pandemic may represent the very basis for the implementation of prospective on-site training and overall scientific update of future urologists.

Disposable versus Reusable Ureteroscopes: A Prospective Multicenter Randomized Comparison.

PURPOSE: To compare reusable and disposable flexible ureteroscopes in terms of efficacy and safety for patients undergoing Retrograde Intrarenal Surgery (RIRS). PATIENTS AND METHODS: Patients with a renal stone eligible for RIRS were enrolled in this multicenter, randomized, clinical trial study. Patients were randomized into two groups: group A (90 patients) underwent RIRS with a reusable flexible ureteroscope and group B (90 patients) were treated with a disposable one. RESULTS: The patients' demographics, stone features and pre-operative urine cultures were comparable between the groups. The Stone Free Rates (SFRs) were not significantly different (86.6% and 90.0% for group A and group B, respectively, p=0.11) and the mean cost for each procedure was comparable (2321 € in group A vs 2543 € in group B, p=0.09). However, the days of hospitalization and of antibiotic therapy were higher in group A (p ≤ 0.05). The overall complication rate in group A was 8.8% whilst in group B it was 3.3% (p ≤ 0.05); in particular, group A exhibited a greater number of major complications (Clavien score IIIa-V). The overall postoperative infection rate was 16.6% in group A and 3.3% in group B (p ≤ 0.05). Furthermore, none of the patients in group B developed urosepsis or had a positive blood culture, while 3 patients in group A did (p<0.05). CONCLUSION: The use of disposable ureteroscopes is characterized by significantly lower post-operative complications and infection rates, while having comparable costs and SFRs vis à vis reusable ureteroscopes. Clinical Trial Registration Number: ISRCTN92289221.

Inhibition of mitochondrial fission and iNOS in the dorsal vagal complex protects from overeating and weight gain.

OBJECTIVES: The dorsal vagal complex (DVC) senses insulin and controls glucose homeostasis, feeding behaviour and body weight. Three-days of high-fat diet (HFD) in rats are sufficient to induce insulin resistance in the DVC and impair its ability to regulate feeding behaviour. HFD-feeding is associated with increased dynamin-related protein 1 (Drp1)-dependent mitochondrial fission in the DVC. We investigated the effects that altered Drp1 activity in the DVC has on feeding behaviour. Additionally, we aimed to uncover the molecular events and the neuronal cell populations associated with DVC insulin sensing and resistance. METHODS: Eight-week-old male Sprague Dawley rats received DVC stereotactic surgery for brain infusion to facilitate the localised administration of insulin or viruses to express mutated forms of Drp1 or to knockdown inducible nitric oxide synthase (iNOS) in the NTS of the DVC. High-Fat diet feeding was used to cause insulin resistance and obesity. RESULTS: We showed that Drp1 activation in the DVC increases weight gain in rats and Drp1 inhibition in HFD-fed rats reduced food intake, weight gain and adipose tissue. Rats expressing active Drp1 in the DVC had higher levels of iNOS and knockdown of DVC iNOS in HFD-fed rats led to a reduction of food intake, weight gain and adipose tissue. Finally, inhibiting mitochondrial fission in DVC astrocytes was sufficient to protect rats from HFD-dependent insulin resistance, hyperphagia, weight gain and fat deposition. CONCLUSION: We uncovered new molecular and cellular targets for brain regulation of whole-body metabolism, which could inform new strategies to combat obesity and diabetes.

Mitochondrial Dynamics in the Brain Are Associated With Feeding, Glucose Homeostasis, and Whole-Body Metabolism.

The brain is responsible for maintaining whole-body energy homeostasis by changing energy input and availability. The hypothalamus and dorsal vagal complex (DVC) are the primary sites of metabolic control, able to sense both hormones and nutrients and adapt metabolism accordingly. The mitochondria respond to the level of nutrient availability by fusion or fission to maintain energy homeostasis; however, these processes can be disrupted by metabolic diseases including obesity and type II diabetes (T2D). Mitochondrial dynamics are crucial in the development and maintenance of obesity and T2D, playing a role in the control of glucose homeostasis and whole-body metabolism across neurons and glia in the hypothalamus and DVC.

Clinical and molecular determinants of extrahepatic disease progression in patients with metastatic colorectal cancer with liver-limited metastases deemed initially unresectable.

BACKGROUND: No tools to predict the probability of extrahepatic disease progression (ePD) of initially unresectable, liver-limited metastatic colorectal cancer (mCRC) are currently available. To estimate the likelihood to develop ePD and to identify clinical and molecular factors that could predict extrahepatic progression-free survival (ePFS), we conducted an observational, retrospective, multicentre cohort study. METHODS: We retrospectively identified a cohort of 225 patients with initially unresectable liver-limited disease (LLD), treated from January 2004 to December 2017 with first-line doublets or triplet plus a biological agent at two Italian institutions. RESULTS: 173 (77%) patients experienced ePD which occurred within 1, 2 or 3 years from the diagnosis of mCRC in 15%, 49% and 66% of patients, respectively. Globally, 164 (73%) patients underwent a liver resection at some point of their disease history, and 54 (33%) of them underwent a subsequent locoregional treatment. Age > 70 years, locoregional nodal involvement at diagnosis of colorectal cancer and ≥4 liver metastases were significantly associated with higher risk of ePD while liver resections were associated with reduced risk of ePD. In the multivariable model, number of liver metastases (subdistribution HR, SHR 1.63, 95% CI 1.12 to 2.36; p = 0.01) and liver resections (SHR 0.43, 95% CI 0.29 to 0.63; p = 0.001) were still associated with ePD. Number of liver metastases < 4, no nodal involvement at diagnosis and liver resections were also associated with prolonged ePFS. CONCLUSIONS: The identified clinical factors could help physicians in personalising the intensity and aggressiveness of liver-directed treatments in patients with mCRC with initially unresectable LLD.

Dynamin-Related Protein 1-Dependent Mitochondrial Fission Changes in the Dorsal Vagal Complex Regulate Insulin Action.

Mitochondria undergo dynamic changes to maintain function in eukaryotic cells. Insulin action in parallel regulates glucose homeostasis, but whether specific changes in mitochondrial dynamics alter insulin action and glucose homeostasis remains elusive. Here, we report that high-fat feeding in rodents incurred adaptive dynamic changes in mitochondria through an increase in mitochondrial fission in parallel to an activation of dynamin-related protein 1 (Drp1) in the dorsal vagal complex (DVC) of the brain. Direct inhibition of Drp1 negated high-fat-feeding-induced mitochondrial fission, endoplasmic reticulum (ER) stress, and insulin resistance in the DVC and subsequently restored hepatic glucose production regulation. Conversely, molecular activation of DVC Drp1 in healthy rodents was sufficient to induce DVC mitochondrial fission, ER stress, and insulin resistance. Together, these data illustrate that Drp1-dependent mitochondrial fission changes in the DVC regulate insulin action and suggest that targeting the Drp1-mitochondrial-dependent pathway in the brain may have therapeutic potential in insulin resistance.

Inhibition of glycine transporter-1 in the dorsal vagal complex improves metabolic homeostasis in diabetes and obesity.

Impaired glucose homeostasis and energy balance are integral to the pathophysiology of diabetes and obesity. Here we show that administration of a glycine transporter 1 (GlyT1) inhibitor, or molecular GlyT1 knockdown, in the dorsal vagal complex (DVC) suppresses glucose production, increases glucose tolerance and reduces food intake and body weight gain in healthy, obese and diabetic rats. These findings provide proof of concept that GlyT1 inhibition in the brain improves glucose and energy homeostasis. Considering the clinical safety and efficacy of GlyT1 inhibitors in raising glycine levels in clinical trials for schizophrenia, we propose that GlyT1 inhibitors have the potential to be repurposed as a treatment of both obesity and diabetes.

Activation of Short and Long Chain Fatty Acid Sensing Machinery in the Ileum Lowers Glucose Production in Vivo.

Evidence continues to emerge detailing the myriad of ways the gut microbiota influences host energy homeostasis. Among the potential mechanisms, short chain fatty acids (SCFAs), the byproducts of microbial fermentation of dietary fibers, exhibit correlative beneficial metabolic effects in humans and rodents, including improvements in glucose homeostasis. The underlying mechanisms, however, remain elusive. We here report that one of the main bacterially produced SCFAs, propionate, activates ileal mucosal free fatty acid receptor 2 to trigger a negative feedback pathway to lower hepatic glucose production in healthy rats in vivo We further demonstrate that an ileal glucagon-like peptide-1 receptor-dependent neuronal network is necessary for ileal propionate and long chain fatty acid sensing to regulate glucose homeostasis. These findings highlight the potential to manipulate fatty acid sensing machinery in the ileum to regulate glucose homeostasis.

Glucagon signalling in the dorsal vagal complex is sufficient and necessary for high-protein feeding to regulate glucose homeostasis in vivo.

High-protein feeding acutely lowers postprandial glucose concentration compared to low-protein feeding, despite a dichotomous rise of circulating glucagon levels. The physiological role of this glucagon rise has been largely overlooked. We here first report that glucagon signalling in the dorsal vagal complex (DVC) of the brain is sufficient to lower glucose production by activating a Gcgr-PKA-ERK-KATP channel signalling cascade in the DVC of rats in vivo. We further demonstrate that direct blockade of DVC Gcgr signalling negates the acute ability of high- vs. low-protein feeding to reduce plasma glucose concentration, indicating that the elevated circulating glucagon during high-protein feeding acts in the brain to lower plasma glucose levels. These data revise the physiological role of glucagon and argue that brain glucagon signalling contributes to glucose homeostasis during dietary protein intake.

Resveratrol activates duodenal Sirt1 to reverse insulin resistance in rats through a neuronal network.

Resveratrol improves insulin sensitivity and lowers hepatic glucose production (HGP) in rat models of obesity and diabetes, but the underlying mechanisms for these antidiabetic effects remain elusive. One process that is considered a key feature of resveratrol action is the activation of the nicotinamide adenine dinucleotide (NAD(+))-dependent deacetylase sirtuin 1 (SIRT1) in various tissues. However, the low bioavailability of resveratrol raises questions about whether the antidiabetic effects of oral resveratrol can act directly on these tissues. We show here that acute intraduodenal infusion of resveratrol reversed a 3 d high fat diet (HFD)-induced reduction in duodenal-mucosal Sirt1 protein levels while also enhancing insulin sensitivity and lowering HGP. Further, we found that duodenum-specific knockdown of Sirt1 expression for 14 d was sufficient to induce hepatic insulin resistance in rats fed normal chow. We also found that the glucoregulatory role of duodenally acting resveratrol required activation of Sirt1 and AMP-activated protein kinase (Ampk) in this tissue to initiate a gut-brain-liver neuronal axis that improved hypothalamic insulin sensitivity and in turn, reduced HGP. In addition to the effects of duodenally acting resveratrol in an acute 3 d HFD-fed model of insulin resistance, we also found that short-term infusion of resveratrol into the duodenum lowered HGP in two other rat models of insulin resistance--a 28 d HFD-induced model of obesity and a nicotinamide (NA)-streptozotocin (STZ)-HFD-induced model of mild type 2 diabetes. Together, these studies highlight the therapeutic relevance of targeting duodenal SIRT1 to reverse insulin resistance and improve glucose homeostasis in obesity and diabetes.

Metformin activates a duodenal Ampk-dependent pathway to lower hepatic glucose production in rats.

Metformin is a first-line therapeutic option for the treatment of type 2 diabetes, even though its underlying mechanisms of action are relatively unclear. Metformin lowers blood glucose levels by inhibiting hepatic glucose production (HGP), an effect originally postulated to be due to a hepatic AMP-activated protein kinase (AMPK)-dependent mechanism. However, studies have questioned the contribution of hepatic AMPK to the effects of metformin on lowering hyperglycemia, and a gut-brain-liver axis that mediates intestinal nutrient- and hormone-induced lowering of HGP has been identified. Thus, it is possible that metformin affects HGP through this inter-organ crosstalk. Here we show that intraduodenal infusion of metformin for 50 min activated duodenal mucosal Ampk and lowered HGP in a rat 3 d high fat diet (HFD)-induced model of insulin resistance. Inhibition of duodenal Ampk negated the HGP-lowering effect of intraduodenal metformin, and both duodenal glucagon-like peptide-1 receptor (Glp-1r)-protein kinase A (Pka) signaling and a neuronal-mediated gut-brain-liver pathway were required for metformin to lower HGP. Preabsorptive metformin also lowered HGP in rat models of 28 d HFD-induced obesity and insulin resistance and nicotinamide (NA)-streptozotocin (STZ)-HFD-induced type 2 diabetes. In an unclamped setting, inhibition of duodenal Ampk reduced the glucose-lowering effects of a bolus metformin treatment in rat models of diabetes. These findings show that, in rat models of both obesity and diabetes, metformin activates a previously unappreciated duodenal Ampk-dependent pathway to lower HGP and plasma glucose levels.

A fatty acid-dependent hypothalamic-DVC neurocircuitry that regulates hepatic secretion of triglyceride-rich lipoproteins.

The brain emerges as a regulator of hepatic triglyceride-rich very-low-density lipoproteins (VLDL-TG). The neurocircuitry involved as well as the ability of fatty acids to trigger a neuronal network to regulate VLDL-TG remain unknown. Here we demonstrate that infusion of oleic acid into the mediobasal hypothalamus (MBH) activates a MBH PKC-δ→KATP-channel signalling axis to suppress VLDL-TG secretion in rats. Both NMDA receptor-mediated transmissions in the dorsal vagal complex (DVC) and hepatic innervation are required for lowering VLDL-TG, illustrating a MBH-DVC-hepatic vagal neurocircuitry that mediates MBH fatty acid sensing. High-fat diet (HFD)-feeding elevates plasma TG and VLDL-TG secretion and abolishes MBH oleic acid sensing to lower VLDL-TG. Importantly, HFD-induced dysregulation is restored with direct activation of either MBH PKC-δ or KATP-channels via the hepatic vagus. Thus, targeting a fatty acid sensing-dependent hypothalamic-DVC neurocircuitry may have therapeutic potential to lower hepatic VLDL-TG and restore lipid homeostasis in obesity and diabetes.

Insulin action in the hypothalamus and dorsal vagal complex.

Insulin resistance is a hallmark feature of type 2 diabetes and obesity. In addition to the classical view that insulin resistance in the liver, muscle and fat disrupts glucose homeostasis, studies in the past decade have illustrated that insulin resistance in the hypothalamus dysregulates hepatic glucose production and food intake, leading to type 2 diabetes and obesity. This invited review argues that in addition to the hypothalamus, insulin signalling in the dorsal vagal complex regulates hepatic glucose production and food intake. A thorough understanding of the physiological and pathophysiological mechanisms of insulin action in the hypothalamus and dorsal vagal complex is necessary in order to identify therapeutic targets for obesity and type 2 diabetes.