GHSR in a Subset of GABA Neurons Controls Food Deprivation-Induced Hyperphagia in Male Mice.

The growth hormone secretagogue receptor (GHSR), primarily known as the receptor for the hunger hormone ghrelin, potently controls food intake, yet the specific Ghsr-expressing cells mediating the orexigenic effects of this receptor remain incompletely characterized. Since Ghsr is expressed in gamma-aminobutyric acid (GABA)-producing neurons, we sought to investigate whether the selective expression of Ghsr in a subset of GABA neurons is sufficient to mediate GHSR's effects on feeding. First, we crossed mice that express a tamoxifen-dependent Cre recombinase in the subset of GABA neurons that express glutamic acid decarboxylase 2 (Gad2) enzyme (Gad2-CreER mice) with reporter mice, and found that ghrelin mainly targets a subset of Gad2-expressing neurons located in the hypothalamic arcuate nucleus (ARH) and that is predominantly segregated from Agouti-related protein (AgRP)-expressing neurons. Analysis of various single-cell RNA-sequencing datasets further corroborated that the primary subset of cells coexpressing Gad2 and Ghsr in the mouse brain are non-AgRP ARH neurons. Next, we crossed Gad2-CreER mice with reactivable GHSR-deficient mice to generate mice expressing Ghsr only in Gad2-expressing neurons (Gad2-GHSR mice). We found that ghrelin treatment induced the expression of the marker of transcriptional activation c-Fos in the ARH of Gad2-GHSR mice, yet failed to induce food intake. In contrast, food deprivation-induced refeeding was higher in Gad2-GHSR mice than in GHSR-deficient mice and similar to wild-type mice, suggesting that ghrelin-independent roles of GHSR in a subset of GABA neurons is sufficient for eliciting full compensatory hyperphagia in mice.

Verapamil and Alzheimer's Disease: Past, Present, and Future.

Verapamil is a phenylalkylamine class calcium channel blocker that for half a century has been used for the treatment of cardiovascular diseases. Nowadays, verapamil is also considered as a drug option for the treatment of several neurological and psychiatric disorders, such as cluster headache, bipolar disorders, epilepsy, and neurodegenerative diseases. Here, we review insights into the potential preventive and therapeutic role of verapamil on Alzheimer's disease (AD) based on limited experimental and clinical data. Pharmacological studies have shown that verapamil has a wide therapeutic spectrum, including antihypertensive, anti-inflammatory, and antioxidative effects, regulation of the blood-brain barrier function, due to its effect on P-glycoprotein, as well as adjustment of cellular calcium homeostasis, which may result in the delay of AD onset or ameliorate the symptoms of patients. However, the majority of the AD individuals are on polypharmacotherapy, and the interactions between verapamil and other drugs need to be considered. Therefore, for an appropriate and successful AD treatment, a personalized approach is more than necessary. A well-known narrow pharmacological window of verapamil efficacy may hinder this approach. It is therefore important to note that the verapamil efficacy may be conditioned by different factors. The onset, grade, and brain distribution of AD pathological hallmarks, the time-sequential appearances of AD-related cognitive and behavioral dysfunction, the chronobiologic and gender impact on calcium homeostasis and AD pathogenesis may somehow be influencing that success. In the future, such insights will be crucial for testing the validity of verapamil treatment on animal models of AD and clinical approaches.

Metyrapone effects on systemic and cerebral energy metabolism.

Metyrapone is a cytochrome P(450) inhibitor that protects against ischemia- and excitotoxicity-induced brain damages in rodents. This study examines whether metyrapone would act on energy metabolism in a manner congruent with its neuroprotective effect. In a first investigation, the rats instrumented with telemetric devices measuring abdominal temperature, received i.p. injection of either metyrapone or saline. One hour after injection, their blood and hippocampus were sampled. Hippocampus metabolite concentrations were measured using (1)H high-resolution magic angle spinning-magnetic resonance spectroscopy ((1)H HRMAS-MRS). The hippocampus levels in phosphorylated mammalian target of rapamycin (mTOR) and adenosine monophosphate-activated protein kinase (AMPK) were measured by Western Blot analysis and those of c-fos and HSP70-2 mRNA were quantified by RT-PCR. In a second investigation, the rats received the same treatment and were sacrificed 1h after. The functioning of mitochondria was immediately studied on their whole brain. Metyrapone provoked a slight hypothermia which was correlated to the increase in blood glucose concentration. Metyrapone also increased blood lactate concentrations without modifying hippocampus lactate content. In the hippocampus, metyrapone decreased γ-aminobutyric acid (GABA) and glutamate levels but increased glutamine and N-acetyl-aspartate contents (NAA). Phosphorylated mTOR and AMPK and the c-fos and HSP70-2 mRNA levels were similar between treatment groups. Metyrapone did not modify blood corticosterone levels. Mitochondrial oxygen consumption was similar in both groups whatever the substrate used. These metabolic modifications, which take place without modifying blood glucocorticoid levels, are consistent with the neuroprotective properties of metyrapone as demonstrated in animal models.

The relationship between locomotion and heat tolerance in heat exposed rats.

Low spontaneous locomotor activity (SA) represents a thermoregulatory behaviour that aims at improving heat tolerance. However, high SA is observed during heat exposure. We hypothesized that high SA could be associated to brain dysfunction. Eighty male Sprague-Dawley rats were heat exposed for 90-min under a continuous assessment of SA and abdominal temperature (T(abd)) using telemetry. The time course analysis showed two SA peaks. The first one was related to exposure to novel environment, the second to heat. The maximal SA level reached in the second peak served to distribute the rats into three groups (LOW, MED and HIGH). In each SA pattern group, heat tolerance was estimated from T(abd) values. At the end of heat exposure, frontal cortex activation was assessed using c-fos, Hsp70 and IkappaBalpha mRNA expressions. The LOW rats exhibited the lowest T(abd), a slight increase in c-fos and Hsp70 mRNA expressions and a robust increase in IkappaBalpha mRNA expression. The HIGH rats exhibited the highest T(abd) and a robust increase in c-fos and Hsp70 mRNA expressions without any change in IkappaBalpha mRNA expression. The c-fos and Hsp70 mRNA expressions were positively correlated to the highest SA levels occurring 45 min before sacrifice, suggesting that high SA and frontal cortex activation are related. In conclusion, high SA is associated to decreased heat tolerance and frontal cortex activation. It may represent a marker of inadequate stress reaction.

Immune function during and after 60 min of moderate exercise wearing protective clothing.

INTRODUCTION: As exercise while wearing protective clothing exacerbates body heat storage compared to exercise in the heat, and as exercise alters immune responses, it appeared worthwhile to examine immune and stress responses while wearing protective clothing during moderate exercise. METHODS: Eight subjects completed two bouts of exercise at 45% Vo2(max) in a thermoneutral environment: once while wearing shorts only (Control trial, CON) and again while wearing protective clothing (PRO). Venous blood samples were taken to analyze TNF-alpha mRNA by RT-PCR in LPS stimulated blood, plasma catecholamines, and cortisol. Blood cell count was analyzed by flow cytometry. Rectal temperature (T(re)) was monitored continuously. RESULTS: Exercise with PRO resulted in significantly greater increases in T(re) (39.2 +/- 0.2 degrees C in PRO vs. 38.0 +/- 0.1 degrees C in CON) and plasma epinephrine and norepinephrine (+70% and 150%, respectively). Plasma cortisol increased only at the end of PRO exercise (+33%). Leukocyte and lymphocyte cell count was 14% and 18% higher, respectively, but there were no significant changes in T cytotoxic and NK cell counts compared to the CON trial. Only T helper lymphocyte count was lower (-29%). During both exercise trials, T helper lymphocytes were significantly decreased at the end of exercise and recovery. With or without protective clothing, exercise was associated with an inhibition of TNF- alpha expression in stimulated monocytes (approximately -50% at min 20 and 40, and approximately -30% at min 60). DISCUSSION: Protective clothing wearing induces significant thermal challenge during exercise. The inhibition of TNF-alpha appears to be mediated primarily by exercise and not the added thermal load associated with protective clothing.

Effects of passive hyperthermia versus exercise-induced hyperthermia on immune responses: hormonal implications.

UNLABELLED: Different stress hormones are released during prolonged exercise and passive hyperthermia. We hypothesized that these different hormonal responses could contribute to the different changes in the immune response during these two challenges. METHODS: Eight subjects completed three trials in a randomized order. In the control trial (C), the subjects remained in a sitting posture for three hours in thermoneutral conditions. In the exercise hyperthermia trial (E), they exercised for two hours on a treadmill at 65% max in thermoneutral conditions, followed by 1-h recovery in thermoneutral conditions; in the passive hyperthermia trial (PH), the subjects remained in a semi-recumbent position in a climatic chamber for two hours in hot conditions, followed by 1-h recovery in thermoneutral conditions. During the E and PH trials, wind speed and thermal conditions were modulated to reach a rectal temperature (Tre) of 38.5 degrees C at 60 min and 39 degrees C at 120 min. The subjects did not drink during the experiments. Blood samples (10 mL) were taken at 0, 60, 120 and 180 min of each trial. The total white cell count and its subsets were measured; plasma catecholamines, cortisol and prolactin were assayed. In a whole blood assay, blood leukocytes were stimulated by lipopolysaccharide (LPS) or phytohemagglutinin (PHA) for 24 and 48 hours, respectively. Cytokines, such as TNF-alpha, IL-10 and INF-gamma were measured in the culture supernatant. RESULTS: The plasma levels of catecholamines were increased only during E, prolactin was increased only during PH, and cortisol was increased in both E and PH. Only the exercise caused a mobilization of blood leukocytes and leukocyte subsets. The INF-gamma and TNF-alpha production by PHA- and LPS-stimulated blood, respectively, were inhibited in a substantial way in both E and PH compared to control when Tre reached 39 degrees C. Only LPS-induced IL-10 production was enhanced during the exercise. The effects of the challenges were increased with 39 degrees C compared to 38.5 degrees C. CONCLUSIONS: Catecholamines play a major role in the mobilization of immunocompetent cells and the production of IL-10 during exercise. Prolactin and catecholamines have adverse role on the immune response, whereas cortisol exerts similar effects during both trials. The consequence could be a protection against inflammatory overshooting.

Effect of glucocorticoid depletion on heat-induced Hsp70, IL-1beta and TNF-alpha gene expression.

When exposed to heat, conscious naive rats may develop lethal heatstroke, depending on heat load, i.e., time spent at high body core temperature. The occurrence of heatstroke was hypothesized to result from a defective glucocorticoid secretion related to altered heat-stress responses. We thus investigated the potential involvement of glucocorticoids in heat tolerance and its consequences on physiological responses, heat shock protein 70 (Hsp70), and cytokine mRNA expressions. Two hours before heat exposure, the animals were injected either with metyrapone, an inhibitor of corticosterone synthesis, or with its vehicle. Heat exposure lasted for 15, 30, 45 or 60 min. Thereafter, the rats were distributed into three groups according to their heat load: null, moderate (without any lethal risk) and intense (with lethal risk). Physiological responses were evaluated with colonic temperature, plasma lactate and hematocrit. Brain responses were assessed in frontal cortex through Hsp70, interleukin-1beta (IL-1beta) and tumor necrosis factor-alpha (TNF-alpha) mRNA expressions. The animals with a severe heat load exhibited a high hematocrit, increased plasma lactate level and enhanced brain IL-1beta and Hsp70 mRNA expressions. Independent of the heat load, Metyrapone rats showed the same thermophysiological responses and IL-1beta and Hsp70 mRNA expressions when compared with vehicle rats. However, the Metyrapone rats experiencing an intense heat load exhibited an increased TNF-alpha mRNA expression. In conclusion, these data (i) confirm that heat load is important in the calibration of the risk attached to heat exposure; and (ii) suggest that corticosterone synthesis inhibition may favor TNF-alpha mRNA expression without any effect on Hsp70 mRNA expression.

Antiapoptotic cytokines in combination with pegfilgrastim soon after irradiation mitigates myelosuppression in nonhuman primates exposed to high irradiation dose.

OBJECTIVE: Preservation of hematopoietic stem and progenitor cells from early radiation-induced apoptosis is the rationale for emergency antiapoptotic cytokine therapy (EACK) after radiation accidents. This strategy is based on the combination of stem cell factor + Flt3-ligand + thrombopoietin + interleukin 3 (SFT3). The long-term safety and efficacy of EACK in managing severe radiation exposure were evaluated. MATERIAL AND METHODS: Early administration of SFT3 + pegfilgrastim was assessed in 7-Gy gamma total body-irradiated (TBI) monkeys. Efficiency of delayed administration was also addressed after 5-Gy TBI. RESULTS: Here we showed that a single, intravenous injection of SFT3 2 hours after 7-Gy TBI reduced the period of thrombocytopenia (platelet count <20 x 10(9)/L: 0.8 +/- 1.5 day vs 23.8 +/- 15.9 days in controls; p < 0.05) and blood transfusion needs. Moreover, addition of pegfilgrastim to SFT3 treatment shortened the period of neutropenia compared with SFT3 and control groups (neutrophil count <0.5 x 10(9)/L: 7 +/- 1.4 days vs 13 +/- 3.2 days and 15.2 +/- 1.5 days; p < 0.05). In both SFT3 groups, bone marrow activity recovered earlier and, in contrast with controls, platelet count returned to baseline values from 250 days after irradiation. Furthermore, delayed (48 hours) single SFT3 administration in 5-Gy irradiated monkeys significantly reduced thrombocytopenia compared to controls. Finally, SFT3 did not increase frequency of total chromosome translocations observed in the blood lymphocytes of controls 1 year after 5 Gy TBI. CONCLUSION: These results suggest the safety and efficacy of EACK in managing severe radiation exposure.

Contraction-induced interleukin-6 transcription in rat slow-type muscle is partly dependent on calcineurin activation.

The present work aimed at determining whether interleukin-6 (IL-6) produced by skeletal muscle during exercise is related, at least partly, to calcineurin activity. Rats were treated with two specific calcineurin inhibitors, cyclosporin A (CsA) and FK506, or vehicle (Vhl); they were then subjected to exhaustive treadmill running. Modulatory Calcineurin-Interacting Protein-1 (MCIP-1) mRNA levels, a reliable indicator of calcineurin activity, and IL-6 mRNA levels were measured by real-time RT-PCR in soleus muscles, and IL-6 protein concentration was measured in the plasma. Because low carbohydrates availability enhances IL-6 transcription through p38 Mitogen Activated Protein Kinase (MAPK) pathway, muscle glycogen content and glycaemia were measured and p38 MAPK phosphorylation was determined in skeletal muscle by western blotting. As expected, exercise induced an increase in IL-6 (P < 0.01) and MCIP-1 mRNA (P < 0.01) in soleus muscle of Vhl rats, and enhanced p38 phosphorylation and plasmatic IL-6 protein (P < 0.05). Calcineurin inhibition did not affect running time, glycemia or soleus glycogen content. CsA administration totally inhibited the exercise-induced increase in MCIP-1 mRNA (P < 0.01), blunted the IL-6 gene transcription related to muscle activity, and suppressed the changes in IL-6 protein in plasma. In addition to its inhibition of calcineurin activity, FK506 administration totally suppressed the exercise-induced IL-6 gene transcription, likely by an inhibition of p38 activation. Taken together, these results demonstrate that in addition to p38 MAPK, increased calcineurin activity is one of the signalling events involved in IL-6 gene transcription.

Kinetic changes of oxidative stress and selenium status in plasma and tissues following burn injury in selenium-deficient and selenium-supplemented rats.

BACKGROUND: This study investigates the relationship between the burn-induced oxidative stress and the selenium status. METHODS: The rats were fed with a selenium-adequate diet or a selenium-depleted diet for 5 weeks, before a third-degree thermal injury was applied to the animals. One group of selenium-depleted animals received injections of sodium selenite after the injury. The selenium status and the oxidative stress parameters were measured for 5 days. RESULTS: The selenium-deficient diet leads to oxidative stress with a high stimulation of the superoxide dismutase activity. After the burn injury, the oxidative stress appears important because the initial selenium status is already impaired and, in all animals, the selenium levels and the antioxidant seleno-dependent glutathione peroxidase (GPx) activity decrease in the plasma and the tissues. A treatment with daily selenium injections is efficient in normalizing selenium levels and restores the GPx activity, but fails to counteract the initial oxidative damages induced by the selenium-deficient diet. CONCLUSIONS: The selenium status before the burn injury is a modulating factor of the burn-induced oxidative stress. A single selenium supplement is not sufficient to counteract these oxidative damages and henceforth combined antioxidant supplementations should be investigated to improve the early treatment of the burn patients.