Limonene and BEZ 235 inhibits growth of COLO-320 and HCT-116 colon cancer cells

D-Limonene is a dietary monoterpene with significant anticancer activity against many cancer types in preclinical and clinical studies. The study is designed to investigate synergistic anticancer effects of limonene and BEZ235 combination in COLO-320 and HCT-116 colon cancer cells. Cells were treated with both the drugs alone and in combination and the effects on cell viability; cell migration and clonogenic potential were examined. Results show that both drugs exhibited dose and time dependant cytotoxicity on the cell lines tested. CompuSyn analysis of the drug combination effects revealed the strong synergistic interaction of the combination. Our results also indicate that COLO-320 cells were more sensitive for anticancer effects of the drugs than HCT-116 cells. The presence of Ras and PI3K mutations in HCT-116 cells could possibly be one of the main reasons for the observed outcome as compared to the wild type expressions of them in COLO-320 cells

Ghrelin does not impact the blunted counterregulatory response to recurrent hypoglycemia in mice

IntroductionRecurrent episodes of insulin-induced hypoglycemia in patients with diabetes mellitus can result in hypoglycemia-associated autonomic failure (HAAF), which is characterized by a compromised response to hypoglycemia by counterregulatory hormones (counterregulatory response; CRR) and hypoglycemia unawareness. HAAF is a leading cause of morbidity in diabetes and often hinders optimal regulation of blood glucose levels. Yet, the molecular pathways underlying HAAF remain incompletely described. We previously reported that in mice, ghrelin is permissive for the usual CRR to insulin-induced hypoglycemia. Here, we tested the hypothesis that attenuated release of ghrelin both results from HAAF and contributes to HAAF.MethodsC57BL/6N mice, ghrelin-knockout (KO) + control mice, and GhIRKO (ghrelin cell-selective insulin receptor knockout) + control mice were randomized to one of three treatment groups: a “Euglycemia” group was injected with saline and remained euglycemic; a 1X hypoglycemia (“1X Hypo”) group underwent a single episode of insulin-induced hypoglycemia; a recurrent hypoglycemia (“Recurrent Hypo”) group underwent repeated episodes of insulin-induced hypoglycemia over five successive days.ResultsRecurrent hypoglycemia exaggerated the reduction in blood glucose (by ~30%) and attenuated the elevations in plasma levels of the CRR hormones glucagon (by 64.5%) and epinephrine (by 52.9%) in C57BL/6N mice compared to a single hypoglycemic episode. Yet, plasma ghrelin was equivalently reduced in “1X Hypo” and “Recurrent Hypo” C57BL/6N mice. Ghrelin-KO mice exhibited neither exaggerated hypoglycemia in response to recurrent hypoglycemia, nor any additional attenuation in CRR hormone levels compared to wild-type littermates. Also, in response to recurrent hypoglycemia, GhIRKO mice exhibited nearly identical blood glucose and plasma CRR hormone levels as littermates with intact insulin receptor expression (floxed-IR mice), despite higher plasma ghrelin in GhIRKO mice.ConclusionsThese data suggest that the usual reduction of plasma ghrelin due to insulin-induced hypoglycemia is unaltered by recurrent hypoglycemia and that ghrelin does not impact blood glucose or the blunted CRR hormone responses during recurrent hypoglycemia

Metabolic insights from a GHSR-A203E mutant mouse model

Objective: Binding of ghrelin to its receptor, growth hormone secretagogue receptor (GHSR), stimulates GH release, induces eating, and increases blood glucose. These processes may also be influenced by constitutive (ghrelin-independent) GHSR activity, as suggested by findings in short people with naturally occurring GHSR-A204E mutations and reduced food intake and blood glucose in rodents administered GHSR inverse agonists, both of which impair constitutive GHSR activity. In this study, we aimed to more fully determine the physiologic relevance of constitutive GHSR activity. Methods: We generated mice with a GHSR mutation that replaces alanine at position 203 with glutamate (GHSR-A203E), which corresponds to the previously described human GHSR-A204E mutation, and used them to conduct ex vivo neuronal electrophysiology and in vivo metabolic assessments. We also measured signaling within COS-7 and HEK293T cells transfected with wild-type GHSR (GHSR-WT) or GHSR-A203E constructs. Results: In COS-7 cells, GHSR-A203E resulted in lower baseline IP3 accumulation than GHSR-WT; ghrelin-induced IP3 accumulation was observed in both constructs. In HEK293T cells co-transfected with voltage-gated CaV2.2 calcium channel complex, GHSR-A203E had no effect on basal CaV2.2 current density while GHSR-WT did; both GHSR-A203E and GHSR-WT inhibited CaV2.2 current in the presence of ghrelin. In cultured hypothalamic neurons from GHSR-A203E and GHSR-deficient mice, native calcium currents were greater than those in neurons from wild-type mice; ghrelin inhibited calcium currents in cultured hypothalamic neurons from both GHSR-A203E and wild-type mice. In brain slices, resting membrane potentials of arcuate NPY neurons from GHSR-A203E mice were hyperpolarized compared to those from wild-type mice; the same percentage of arcuate NPY neurons from GHSR-A203E and wild-type mice depolarized upon ghrelin exposure. The GHSR-A203E mutation did not significantly affect body weight, body length, or femur length in the first ∼6 months of life, yet these parameters were lower in GHSR-A203E mice after 1 year of age. During a 7-d 60% caloric restriction regimen, GHSR-A203E mice lacked the usual marked rise in plasma GH and demonstrated an exaggerated drop in blood glucose. Administered ghrelin also exhibited reduced orexigenic and GH secretagogue efficacies in GHSR-A203E mice. Conclusions: Our data suggest that the A203E mutation ablates constitutive GHSR activity and that constitutive GHSR activity contributes to the native depolarizing conductance of GHSR-expressing arcuate NPY neurons. Although the A203E mutation does not block ghrelin-evoked signaling as assessed using in vitro and ex vivo models, GHSR-A203E mice lack the usual acute food intake response to administered ghrelin in vivo. The GHSR-A203E mutation also blunts GH release, and in aged mice leads to reduced body length and femur length, which are consistent with the short stature of human carriers of the GHSR-A204E mutation.Fil: Torz, Lola J.. Universidad de Copenhagen; DinamarcaFil: Osborne Lawrence, Sherri. Ut Southwestern Medical Center; Estados UnidosFil: Rodriguez, Juan. Ut Southwestern Medical Center; Estados UnidosFil: He, Zhenyan. Ut Southwestern Medical Center; Estados UnidosFil: Cornejo, María Paula. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - La Plata. Instituto Multidisciplinario de Biología Celular. Provincia de Buenos Aires. Gobernación. Comisión de Investigaciones Científicas. Instituto Multidisciplinario de Biología Celular. Universidad Nacional de La Plata. Instituto Multidisciplinario de Biología Celular; ArgentinaFil: Mustafá, Emilio Román. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - La Plata. Instituto Multidisciplinario de Biología Celular. Provincia de Buenos Aires. Gobernación. Comisión de Investigaciones Científicas. Instituto Multidisciplinario de Biología Celular. Universidad Nacional de La Plata. Instituto Multidisciplinario de Biología Celular; ArgentinaFil: Jin, Chunyu. Universidad de Copenhagen; DinamarcaFil: Petersen, Natalia. Universidad de Copenhagen; DinamarcaFil: Hedegaard, Morten A.. Universidad de Copenhagen; DinamarcaFil: Nybo, Maja. Universidad de Copenhagen; DinamarcaFil: Martínez Damonte, Valentina. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - La Plata. Instituto Multidisciplinario de Biología Celular. Provincia de Buenos Aires. Gobernación. Comisión de Investigaciones Científicas. Instituto Multidisciplinario de Biología Celular. Universidad Nacional de La Plata. Instituto Multidisciplinario de Biología Celular; ArgentinaFil: Metzger, Nathan P.. Ut Southwestern Medical Center; Estados UnidosFil: Mani, Bharath K.. Ut Southwestern Medical Center; Estados UnidosFil: Williams, Kevin W.. Ut Southwestern Medical Center; Estados UnidosFil: Raingo, Jesica. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - La Plata. Instituto Multidisciplinario de Biología Celular. Provincia de Buenos Aires. Gobernación. Comisión de Investigaciones Científicas. Instituto Multidisciplinario de Biología Celular. Universidad Nacional de La Plata. Instituto Multidisciplinario de Biología Celular; ArgentinaFil: Perello, Mario. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - La Plata. Instituto Multidisciplinario de Biología Celular. Provincia de Buenos Aires. Gobernación. Comisión de Investigaciones Científicas. Instituto Multidisciplinario de Biología Celular. Universidad Nacional de La Plata. Instituto Multidisciplinario de Biología Celular; ArgentinaFil: Holst, Birgitte. Universidad de Copenhagen; DinamarcaFil: Zigman, Jeffrey M.. Ut Southwestern Medical Center; Estados Unido

Altered ghrelin secretion in mice in response to diet-induced obesity and Roux-en-Y gastric bypass

The current study examined potential mechanisms for altered circulating ghrelin levels observed in diet-induced obesity (DIO) and following weight loss resulting from Roux-en-Y gastric bypass (RYGB). We hypothesized that circulating ghrelin levels were altered in obesity and after weight loss through changes in ghrelin cell responsiveness to physiological cues. We confirmed lower ghrelin levels in DIO mice and demonstrated elevated ghrelin levels in mice 6 weeks post-RYGB. In both DIO and RYGB settings, these changes in ghrelin levels were associated with altered ghrelin cell responsiveness to two key physiological modulators of ghrelin secretion – glucose and norepinephrine. In DIO mice, increases in ghrelin cell density within both the stomach and duodenum and in somatostatin-immunoreactive D cell density in the duodenum were observed. Our findings provide new insights into the regulation of ghrelin secretion and its relation to circulating ghrelin within the contexts of obesity and weight loss

Differential Effects of Selective Cyclooxygenase-2 Inhibitors on Vascular Smooth Muscle Ion Channels May Account for Differences in Cardiovascular Risk Profiles

Celecoxib, rofecoxib, and diclofenac are clinically used cyclooxygenase-2 (COX-2) inhibitors, which have been under intense scrutiny because long-term rofecoxib (Vioxx; Merck, Whitehouse Station, NJ) treatment was found to increase the risk of adverse cardiovascular events. A differential risk profile for these drugs has emerged, but the underlying mechanisms have not been fully elucidated. We investigated the effects of celecoxib, rofecoxib, and diclofenac on ionic currents and calcium signaling in vascular smooth muscle cells (VSMCs) using patch-clamp techniques and fura-2 fluorescence and on arterial constriction using pressure myography. Celecoxib, but not rofecoxib or diclofenac, dramatically enhanced KCNQ (Kv7) potassium currents and suppressed L-type voltage-sensitive calcium currents in A7r5 rat aortic smooth muscle cells (native KCNQ currents or overexpressed human KCNQ5 currents) and freshly isolated rat mesenteric artery myocytes. The effects of celecoxib were concentration-dependent within the therapeutic concentration range, and were reversed on washout. Celecoxib, but not rofecoxib, also inhibited calcium responses to vasopressin in A7r5 cells and dilated intact or endothelium-denuded rat mesenteric arteries. A celecoxib analog, 2,5-dimethyl-celecoxib, which does not inhibit COX-2, mimicked celecoxib in its enhancement of vascular KCNQ5 currents, suppression of L-type calcium currents, and vasodilation. We conclude that celecoxib inhibits calcium responses in VSMCs by enhancing KCNQ5 currents and suppressing L-type calcium currents, which ultimately reduces vascular tone. These effects are independent of its COX-2 inhibitory actions and may explain the differential risk of cardiovascular events in patients taking different drugs of this class

Cyber-Attacks in IoT-enabled Cyber-physical Systems

Cyber physical systems (CPS) that are Internet of Things (IoT) enabled might be difficult to secure since security measures designed for general data / value through the development (IT / OT) systems may not work as well in a CPS environment. Consequently, this research provides a two-level ensemble attack detection and attribution framework created for CPS, and more particularly in an industrial control system (ICS). For identifying assaults in unbalanced ICS environments, a decision tree integrated to an unique ensemble deep representation learning model is created at the first extent. An ensemble deep neural network is created for assault features at the second level. Applying actual data collections from the gas pipeline and water treatment system, Findings show that the suggested type is more effective than other competing methods with a similar level of computational complexity

Ghrelin mediates exercise endurance and the feeding response post-exercise

Objective: Exercise training has several well-established health benefits, including many related to body weight, appetite control, and blood glucose homeostasis. However, the molecular mechanisms and, in particular, the hormonal systems that mediate and integrate these beneficial effects are poorly understood. In the current study, we aimed to investigate the role of the hormone ghrelin and its receptor, the growth hormone secretagogue receptor (GHSR; ghrelin receptor), in mediating the effects of exercise on food intake and blood glucose following exercise as well as in regulating exercise endurance capacity. Methods: We used two mouse models of treadmill running to characterize the changes in plasma ghrelin with exercise. We also assessed the role of the ghrelin system to influence food intake and blood glucose after exercise, exercise endurance, and parameters potentially linked to responses to exercise. Mice lacking GHSRs (GHSR-null mice) and wild-type littermates were studied. Results: An acute bout of exercise transiently elevated plasma acyl-ghrelin. Without the action of this increased ghrelin on GHSRs (as in GHSR-null mice), high intensity interval exercise markedly reduced food intake compared to control mice. The effect of exercise to acutely raise blood glucose remained unmodified in GHSR-null mice. Exercise-induced increases in plasma ghrelin positively correlated with endurance capacity, and time to exhaustion was reduced in GHSR-null mice as compared to wild-type littermates. In an effort to mechanistically explain their reduced exercise endurance, exercised GHSR-null mice exhibited an abrogated sympathoadrenal response, lower overall insulin-like growth factor-1 levels, and altered glycogen utilization. Conclusions: Exercise transiently increases plasma ghrelin. GHSR-null mice exhibit decreased food intake following high intensity interval exercise and decreased endurance when submitted to an exercise endurance protocol. These data suggest that an intact ghrelin system limits the capacity of exercise to restrict food intake following exercise, although it enhances exercise endurance. Keywords: GHSR, Ghrelin, Exercise, Treadmill, Endurance, Food intak

The role of ghrelin-responsive mediobasal hypothalamic neurons in mediating feeding responses to fasting

Objective: Ghrelin is a stomach-derived hormone that affects food intake and regulates blood glucose. The best-characterized actions of ghrelin are mediated by its binding to and activation of the growth hormone secretagogue receptor (GHSR; ghrelin receptor). Adequate examination of the identity, function, and relevance of specific subsets of GHSR-expressing neurons has been hampered by the absence of a suitable Cre recombinase (Cre)-expressing mouse line with which to manipulate gene expression in a targeted fashion within GHSR-expressing neurons. The present study aims to characterize the functional significance and neurocircuitry of GHSR-expressing neurons in the mediobasal hypothalamus (MBH), as they relate to ghrelin-induced food intake and fasting-associated rebound hyperphagia, using a novel mouse line in which Cre expression is controlled by the Ghsr promoter. Methods: A Ghsr-IRES-Cre mouse line that expresses Cre directed by the Ghsr promoter was generated. The line was validated by comparing Cre activity in reporter mice to the known brain distribution pattern of GHSR. Next, the requirement of MBH GHSR-expressing neuronal activity in mediating food intake in response to administered ghrelin and in response to fasting was assessed after stereotaxic delivery of inhibitory designer receptor exclusively activated by designer drugs (DREADD) virus to the MBH. In a separate cohort of Ghsr-IRES-Cre mice, stereotaxic delivery of stimulatory DREADD virus to the MBH was performed to assess the sufficiency of MBH GHSR-expressing neuronal activity on food intake. Finally, the distribution of MBH GHSR-expressing neuronal axonal projections was assessed in the DREADD virus-injected animals. Results: The pattern of Cre activity in the Ghsr-IRES-Cre mouse line mostly faithfully reproduced the known GHSR expression pattern. DREADD-assisted inhibition of MBH GHSR neuronal activity robustly suppressed the normal orexigenic response to ghrelin and fasting-associated rebound food intake. DREADD-assisted stimulation of MBH GHSR neuronal activity was sufficient to induce food intake. Axonal projections of GHSR-expressing MBH neurons were observed in a subset of hypothalamic and extra-hypothalamic regions. Conclusions: These results suggest that 1) activation of GHSR-expressing neurons in the MBH is required for the normal feeding responses following both peripheral administration of ghrelin and fasting, 2) activation of MBH GHSR-expressing neurons is sufficient to induce feeding, and 3) axonal projections to a subset of hypothalamic and/or extra-hypothalamic regions likely mediate these responses. The Ghsr-IRES-Cre line should serve as a valuable tool to further our understanding of the functional significance of ghrelin-responsive/GHSR-expressing neurons and the neuronal circuitry within which they act