Islet Brain 1 Protects Insulin Producing Cells against Lipotoxicity.

Chronic intake of saturated free fatty acids is associated with diabetes and may contribute to the impairment of functional beta cell mass. Mitogen activated protein kinase 8 interacting protein 1 also called islet brain 1 (IB1) is a candidate gene for diabetes that is required for beta cell survival and glucose-induced insulin secretion (GSIS). In this study we investigated whether IB1 expression is required for preserving beta cell survival and function in response to palmitate. Chronic exposure of MIN6 and isolated rat islets cells to palmitate led to reduction of the IB1 mRNA and protein content. Diminution of IB1 mRNA and protein level relied on the inducible cAMP early repressor activity and proteasome-mediated degradation, respectively. Suppression of IB1 level mimicked the harmful effects of palmitate on the beta cell survival and GSIS. Conversely, ectopic expression of IB1 counteracted the deleterious effects of palmitate on the beta cell survival and insulin secretion. These findings highlight the importance in preserving the IB1 content for protecting beta cell against lipotoxicity in diabetes.

Most of the Abundant Protein Fractions of Embryonic Cerebrospinal Fluid are Produced Out of the Brain Anlagen

The microenvironment of the central nervous system is important for neuronal function and development. During the early stages of embryo development the cephalic vesicles are filled by embryonic cerebrospinal fluid, a complex fluid containing different protein fractions, which contributes to the regulation of the survival, proliferation and neurogenesis of neuroectodermal stem cells. The protein content of embryonic cerebrospinal fluid from chick and rat embryos at the start of neurogenesis has already been determined. Most of the identified gene products are thought to be involved in the regulation of developmental processes during embryogenesis. However, due to the crucial roles played by embryonic cerebrospinal fluid during brain development, the embryological origin of the gene products it contains remains an intriguing question. According to the literature most of these products are synthesised in embryonic tissues other than the neuroepithelium. In this study we examined the embryological origin of the most abundant embryonic cerebrospinal fluid protein fractions by means of slot-blot analysis and by using several different embryonic and extraembryonic protein extracts, immunodetected with polyclonal antibodies. This first attempt to elucidate their origin is not based on the proteins identified by proteomic methods, but rather on crude protein fractions detected by SDS-PAGE analysis and to which polyclonal antibodies were specifically generated. Despite some of the limitations of this study, i.e. that one protein fraction may contain more than one gene product, and that a specific gene product may be contained in different protein fractions depending on post-translational modifications, our results show that most of the analysed protein fractions are not produced by the cephalic neuroectoderm but are rather stored in the egg reservoir; furthermore, few are produced by embryo tissues, thus indicating that they must be transported from their production or storage sites to the cephalic cavities, most probably via embryonic serum. These results raise the question as to whether the transfer of proteins from these two embryo compartments is regulated at this early developmental stage.

Rapid eye movement (REM) sleep deprivation reduces rat frontal cortex acetylcholinesterase (EC 3.1.1.7) activity

Rapid eye movement (REM) sleep deprivation induces several behavioral changes. Among these, a decrease in yawning behavior produced by low doses of cholinergic agonists is observed which indicates a change in brain cholinergic neurotransmission after REM sleep deprivation. Acetylcholinesterase (Achase) controls acetylcholine (Ach) availability in the synaptic cleft. Therefore, altered Achase activity may lead to a change in Ach availability at the receptor level which, in turn, may result in modification of cholinergic neurotransmission. To determine if REM sleep deprivation would change the activity of Achase, male Wistar rats, 3 months old, weighing 250-300 g, were deprived of REM sleep for 96 h by the flower-pot technique (N = 12). Two additional groups, a home-cage control (N = 6) and a large platform control (N = 6), were also used. Achase was measured in the frontal cortex using two different methods to obtain the enzyme activity. One method consisted of the obtention of total (900 g supernatant), membrane-bound (100,000 g pellet) and soluble (100,000 g supernatant) Achase, and the other method consisted of the obtention of a fraction (40,000 g pellet) enriched in synaptic membrane-bound enzyme. In both preparations, REM sleep deprivation induced a significant decrease in rat frontal cortex Achase activity when compared to both home-cage and large platform controls. REM sleep deprivation induced a significant decrease of 16% in the membrane-bound Achase activity (nmol thiocholine formed min-1 mg protein-1) in the 100,000 g pellet enzyme preparation (home-cage group 152.1 ± 5.7, large platform group 152.7 ± 24.9 and REM sleep-deprived group 127.9 ± 13.8). There was no difference in the soluble enzyme activity. REM sleep deprivation also induced a significant decrease of 20% in the enriched synaptic membrane-bound Achase activity (home-cage group 126.4 ± 21.5, large platform group 127.8 ± 20.4, REM sleep-deprived group 102.8 ± 14.2). Our results suggest that REM sleep deprivation changes Ach availability at the level of its receptors through a decrease in Achase activity

Volatile cardioplegia: fast normothermic cardiac arrest induction and recovery with halothane in isolated rat hearts

To study the effect of halothane as a cardioplegic agent, ten Wistar rats were anesthetized by ether inhalation and their hearts were perfused in a Langendorff system with Krebs-Henseleit solution (36oC; 90 cm H2O pressure). After a 15-min period for stabilization the control values for heart rate, force (T), dT/dt and coronary flow were recorded and a halothane-enriched solution (same temperature and pressure) was perfused until cardiac arrest was obtained. The same Krebs-Henseleit solution was reperfused again and the parameters studied were recorded after 1, 3, 5, 10, 20 and 30 min. Cardiac arrest occurred in all hearts during the first two min of perfusion with halothane-bubbled solution. One minute after reperfusion without halothane, the following parameters reported in terms of control values were obtained: 90.5% of control heart rate (266.9 ± 43.4 to 231.5 ± 71.0 bpm), 20.2% of the force (1.83 ± 0.28 to 0.37 ± 0.25 g), 19.8% of dT/dt (46.0 ± 7.0 to 9.3 ± 6.0 g/s) and 90.8% of coronary flow (9.9 ± 1.5 to 9.4 ± 1.5 ml/min). After 3 min of perfusion they changed to 99.0% heart rate (261.0 ± 48.2), 98.9% force (1.81 ± 0.33), 98.6 dT/dt (45.0 ± 8.2) and 94.8% coronary flow (9.3 ± 1.4). At 5 min 100.8% (267.0 ± 40.6) heart rate, 105.0% (1.92 ± 0.29) force and 104.4% (48.2 ± 7.2) dT/dt were recorded and maintained without significant differences (P>0.01) until the end of the experiment. These data demonstrate that volatile cardioplegia with halothane is an effective technique for fast induction of and prompt recovery from normothermic cardiac arrest of the rat heart

Effect of angiotensin-(1-7) on reperfusion arrhythmias in isolated rat hearts

There is increasing evidence that angiotensin-(1-7) (Ang-(1-7)) is an endogenous biologically active component of the renin-angiotensin system (RAS). In the present study, we investigated the effects of Ang-(1-7) on reperfusion arrhythmias in isolated rat hearts. Isolated rat hearts were perfused with two different media, i.e., Krebs-Ringer (2.52 mM CaCl2) and low-Ca2+ Krebs-Ringer (1.12 mM CaCl2). In hearts perfused with Krebs-Ringer, Ang-(1-7) produced a concentration-dependent (27-210 nM) reduction in coronary flow (25% reduction at highest concentration), while only slight and variable changes in contraction force and heart rate were observed. Under the same conditions, angiotensin II (Ang II; 27 and 70 nM) produced a significant reduction in coronary flow (39% and 48%, respectively) associated with a significant increase in force. A decrease in heart rate was also observed. In low-Ca2+ Krebs-Ringer solution, perfusion with Ang-(1-7) or Ang II at 27 nM concentration produced similar changes in coronary flow, contraction force and heart rate. In isolated hearts perfused with normal Krebs-Ringer, Ang-(1-7) produced a significant enhancement of reperfusion arrhythmias revealed by an increase in the incidence and duration of ventricular tachycardia and ventricular fibrillation (more than 30-min duration). The facilitation of reperfusion arrhythmias by Ang-(1-7) was associated with an increase in the magnitude of the decreased force usually observed during the post-ischemic period. The effects of Ang-(1-7) were abolished in isolated rat hearts perfused with low-Ca2+ Krebs-Ringer. The effect of Ang II (27 nM) was similar but less pronounced than that of Ang-(1-7) at the same concentration. These results indicate that the heart is a site of action for Ang-(1-7) and suggest that this heptapeptide may be involved in the mediation of the cardiac effects of the RAS

Inflammatory response of the spinal cord to multiple episodes of blood-brain barrier disruption and toxic demyelination in Wistar rats

Multiple episodes of blood-brain barrier disruption were induced by sequential intraspinal injections of ethidium bromide. In addition to the barrier disruption, there was toxic demyelination and exposure of myelin components to the immune system. Twenty-seven 3-month-old Wistar rats received 2, 3 or 4 injections of 1 µl of either 0.1% ethidium bromide in normal saline (19 rats) or 0.9% saline (8 rats) at different levels of the spinal cord. The time intervals between the injections ranged from 28 to 42 days. Ten days after the last injection, all rats were perfused with 2.5% glutaraldehyde. The spinal sections were evaluated macroscopically and by light and transmission electron microscopy. All the lesions demonstrated a mononuclear phagocytic infiltrate apparently removing myelin. Lymphocytes were not conspicuous and were found in only 34% of the lesions. No perivascular cuffings were detected. In older lesions (38 days and older) they were found only within Virchow-Robin spaces. This result suggests that multiple blood-brain barrier disruptions with demyelination and exposure of myelin components to the immune system were not sufficient to induce an immune-mediated reaction in the central nervous system.

Gender differences in the activities of aspirin-esterases in rat tissues

The activities of aspirin (acetylsalicylic acid)-esterases were measured in several tissues (liver, kidney, adrenal glands, brain and serum) from adult male and female Wistar rats. In males, both aspirin-esterase I (assayed at pH 5.5) and II (assayed at pH 7.4) activities were higher in liver homogenates when compared to females (aspirin-esterase I: males 48.9 ± 4.8 (N = 8) and females 29.3 ± 4.2 (N = 8) nmol of salicylic acid formed min-1 mg protein-1; aspirin-esterase II: males 41.4 ± 4.1 (N = 8) and females 26.1 ± 4.5 (N = 8) nmol of salicylic acid formed min-1 mg protein-1, P<0.001). In serum, enzyme activity was higher in females than in males (aspirin-esterase I: males 0.85 ± 0.06 (N = 6) and females 1.18 ± 0.11 (N = 6) nmol of salicylic acid formed min-1 mg protein-1; aspirin-esterase II: males 1.03 ± 0.13 (N = 6) and females 1.34 ± 0.11 (N = 6) nmol of salicylic acid formed min-1 mg protein-1, P<0.001). In the other tissues assayed, no statistically significant difference between males and females was found. There were no statistically significant differences when the enzymes were assayed in different phases of the estrous cycle in liver and serum. These results show that the differences in aspirin-esterase activity observed between males and females are not due to the estrous cycle. The gender difference obtained in our study may indicate an involvement of gonadal hormones in the control of the hydrolysis of aspirin. This possibility is currently under investigation.

The left ventricular contractility of the rat heart is modulated by changes in flow and a1-adrenoceptor stimulation

Myocardial contractility depends on several mechanisms such as coronary perfusion pressure (CPP) and flow as well as on a1-adrenoceptor stimulation. Both effects occur during the sympathetic stimulation mediated by norepinephrine. Norepinephrine increases force development in the heart and produces vasoconstriction increasing arterial pressure and, in turn, CPP. The contribution of each of these factors to the increase in myocardial performance needs to be clarified. Thus, in the present study we used two protocols: in the first we measured mean arterial pressure, left ventricular pressure and rate of rise of left ventricular pressure development in anesthetized rats (N = 10) submitted to phenylephrine (PE) stimulation before and after propranolol plus atropine treatment. These observations showed that in vivo a1-adrenergic stimulation increases left ventricular-developed pressure (P<0.05) together with arterial blood pressure (P<0.05). In the second protocol, we measured left ventricular isovolumic systolic pressure (ISP) and CPP in Langendorff constant flow-perfused hearts. The hearts (N = 7) were perfused with increasing flow rates under control conditions and PE or PE + nitroprusside (NP). Both CPP and ISP increased (P<0.01) as a function of flow. CPP changes were not affected by drug treatment but ISP increased (P<0.01). The largest ISP increase was obtained with PE + NP treatment (P<0.01). The results suggest that both mechanisms, i.e., direct stimulation of myocardial a1-adrenoceptors and increased flow, increased cardiac performance acting simultaneously and synergistically.

Inhibition of rat microsomal lipid peroxidation by the oral administration of D002

The effect of D002, a defined mixture of higher primary alcohols purified from bee wax, on in vivo and in vitro lipid peroxidation was studied. The extent of lipid peroxidation was measured on the basis of the levels of thiobarbituric acid reactive substances (TBARS). When D002 (5-100 mg/kg body weight) was administered orally to rats for two weeks, a partial inhibition of the in vitro enzymatic and non-enzymatic lipid peroxidation was observed in liver and brain microsomes. Maximal protection (46%) occurred at a dose of 25 mg/kg. D002 behaved differently depending on both the presence of NADPH and the integrity of liver microsomes, which suggests that under conditions where microsomal metabolism was favored the protective effect of D002 was increased. D002 (25 mg/kg) also completely inhibited carbon tetrachloride- and toluene-induced in vivo lipid peroxidation in liver and brain. Also, D002 significantly lowered in a dose-dependent manner the basal level of TBARS in liver (19-40%) and brain (28-44%) microsomes. We conclude that the oral administration of D002 (5, 25 and 100 mg/kg) for two weeks protected rat liver and brain microsomes against microsomal lipid peroxidation in vitro and in vivo. Thus, D002 could be useful as a dietary natural antioxidant supplement. More studies are required before these data can be extrapolated to the recommendation for the use of D002 as a dietary antioxidant supplement for humans.

Induction of Fos protein immunoreactivity by spinal cord contusion

The objective of the present study was to identify neurons in the central nervous system that respond to spinal contusion injury in the rat by monitoring the expression of the nuclear protein encoded by the c-fos gene, an activity-dependent gene, in spinal cord and brainstem regions. Rats were anesthetized with urethane and the injury was produced by dropping a 5-g weight from 20.0 cm onto the exposed dura at the T10-L1 vertebral level (contusion group). The spinal cord was exposed but not lesioned in anesthetized control animals (laminectomy group); intact animals were also subjected to anesthesia (intact control). Behavioral alterations were analyzed by Tarlov/Bohlman scores, 2 h after the procedures and the animals were then perfused for immunocytochemistry. The patterns of Fos-like immunoreactivity (FLI) which were site-specific, reproducible and correlated with spinal laminae that respond predominantly to noxious stimulation or injury: laminae I-II (outer substantia gelatinosa) and X and the nucleus of the intermediolateral cell column. At the brain stem level FLI was detected in the reticular formation, area postrema and solitary tract nucleus of lesioned animals. No Fos staining was detected by immunocytochemistry in the intact control group. However, detection of FLI in the group submitted to anesthesia and surgical procedures, although less intense than in the lesion group, indicated that microtraumas may occur which are not detected by the Tarlov/Bohlman scores. There is both a local and remote effect of a distal contusion on the spinal cord of rats, implicating sensory neurons and centers related to autonomic control in the reaction to this kind of injury.

Oxytocin is a cardiovascular hormone

Oxytocin (OT), a nonapeptide, was the first hormone to have its biological activities established and chemical structure determined. It was believed that OT is released from hypothalamic nerve terminals of the posterior hypophysis into the circulation where it stimulates uterine contractions during parturition, and milk ejection during lactation. However, equivalent concentrations of OT were found in the male hypophysis, and similar stimuli of OT release were determined for both sexes, suggesting other physiological functions. Indeed, recent studies indicate that OT is involved in cognition, tolerance, adaptation and complex sexual and maternal behaviour, as well as in the regulation of cardiovascular functions. It has long been known that OT induces natriuresis and causes a fall in mean arterial pressure, both after acute and chronic treatment, but the mechanism was not clear. The discovery of the natriuretic family shed new light on this matter. Atrial natriuretic peptide (ANP), a potent natriuretic and vasorelaxant hormone, originally isolated from rat atria, has been found at other sites, including the brain. Blood volume expansion causes ANP release that is believed to be important in the induction of natriuresis and diuresis, which in turn act to reduce the increase in blood volume. Neurohypophysectomy totally abolishes the ANP response to volume expansion. This indicates that one of the major hypophyseal peptides is responsible for ANP release. The role of ANP in OT-induced natriuresis was evaluated, and we hypothesized that the cardio-renal effects of OT are mediated by the release of ANP from the heart. To support this hypothesis, we have demonstrated the presence and synthesis of OT receptors in all heart compartments and the vasculature. The functionality of these receptors has been established by the ability of OT to induce ANP release from perfused heart or atrial slices. Furthermore, we have shown that the heart and large vessels like the aorta and vena cava are sites of OT synthesis. Therefore, locally produced OT may have important regulatory functions within the heart and vascular beds. Such functions may include slowing down of the heart or the regulation of local vascular tone.

Angiotensin-(1-7) potentiates the coronary vasodilatatory effect of bradykinin in the isolated rat heart

It has been shown that angiotensin-(1-7) (Ang-(1-7)) infusion potentiates the bradykinin (BK)-induced hypotensive response in conscious rats. The present study was conducted to identify Ang-(1-7)-BK interactions in the isolated rat heart perfused according to the Langendorff technique. Hearts were excised and perfused through the aortic stump under a constant flow with Krebs-Ringer solution and the changes in perfusion pressure and heart contractile force were recorded. Bolus injections of BK (2.5, 5, 10 and 20 ng) produced a dose-dependent hypotensive effect. Ang-(1-7) added to the perfusion solution (2 ng/ml) did not change the perfusion pressure or the contractile force but doubled the hypotensive effect of the lower doses of BK. The BK-potentiating Ang-(1-7) activity was blocked by pretreatment with indomethacin (5 mg/kg, ip) or L-NAME (30 mg/kg, ip). The Ang-(1-7) antagonist A-779 (50 ng/ml in Krebs-Ringer) completely blocked the effect of Ang-(1-7) on BK-induced vasodilation. These data suggest that the potentiation of the BK-induced vasodilation by Ang-(1-7) can be attributed to the release of nitric oxide and vasodilator prostaglandins through an Ang-(1-7) receptor-mediated mechanism.

Brain ischemia alters platelet ATP diphosphohydrolase and 5'-nucleotidase activities in naive and preconditioned rats

The effects of transient forebrain ischemia, reperfusion and ischemic preconditioning on rat blood platelet ATP diphosphohydrolase and 5'-nucleotidase activities were evaluated. Adult Wistar rats were submitted to 2 or 10 min of single ischemic episodes, or to 10 min of ischemia 1 day after a 2-min ischemic episode (ischemic preconditioning) by the four-vessel occlusion method. Rats submitted to single ischemic insults were reperfused for 60 min and for 1, 2, 5, 10 and 30 days after ischemia; preconditioned rats were reperfused for 60 min 1 and 2 days after the long ischemic episode. Brain ischemia (2 or 10 min) inhibited ATP and ADP hydrolysis by platelet ATP diphosphohydrolase. On the other hand, AMP hydrolysis by 5'-nucleotidase was increased after 2, but not 10, min of ischemia. Ischemic preconditioning followed by 10 min of ischemia caused activation of both enzymes. Variable periods of reperfusion distinctly affected each experimental group. Enzyme activities returned to control levels in the 2-min group. However, the decrease in ATP diphosphohydrolase activity was maintained up to 30 days of reperfusion after 10-min ischemia. 5'-Nucleotidase activity was decreased 60 min and 1 day following 10-min ischemia; interestingly, enzymatic activity was increased after 2 and 5 days of reperfusion, and returned to control levels after 10 days. Ischemic preconditioning cancelled the effects of 10-min ischemia on the enzymatic activities. These results indicate that brain ischemia and ischemic preconditioning induce peripheral effects on ecto-enzymes from rat platelets involved in nucleotide metabolism. Thus, ATP, ADP and AMP degradation and probably the generation of adenosine in the circulation may be altered, leading to regulation of microthrombus formation since ADP aggregates platelets and adenosine is an inhibitor of platelet aggregation.

Rat liver responsiveness to gluconeogenic substrates during insulin-induced hypoglycemia

Hepatic responsiveness to gluconeogenic substrates during insulin-induced hypoglycemia was investigated. For this purpose, livers were perfused with a saturating concentration of 2 mM glycerol, 5 mM L-alanine or 5 mM L-glutamine as gluconeogenic substrates. All experiments were performed 1 h after an ip injection of saline (CN group) or 1 IU/kg of insulin (IN group). The IN group showed higher (P<0.05) hepatic glucose production from glycerol, L-alanine and L-glutamine and higher (P<0.05) production of L-lactate, pyruvate and urea from L-alanine and L-glutamine. In addition, ip injection of 100 mg/kg glycerol, L-alanine and L-glutamine promoted glucose recovery. The results indicate that the hepatic capacity to produce glucose from gluconeogenic precursors was increased during insulin-induced hypoglycemia.

Actions of Crotalus durissus terrificus venom and crotoxin on the isolated rat kidney

Many studies have reported the occurrence of lethal acute renal failure after snakebites. The aim of the present investigation was to determine alterations in renal function produced by Crotalus durissus terrificus venom and crotoxin as well as the histological alterations induced by these venoms. Isolated kidneys from Wistar rats weighing 240 to 280 g were perfused with Krebs-Henseleit solution containing 6 g% of previously dialyzed bovine serum albumin. The effects of Crotalus durissus terrificus venom and crotoxin were studied on glomerular filtration rate (GFR), urinary flow (UF), perfusion pressure (PP) and percentage sodium tubular transport (%TNa+). The infusion of Crotalus durissus terrificus venom (10 µg/ml) and crotoxin (10 µg/ml) increased GFR (control80 = 0.78 ± 0.07, venom80 = 1.1 ± 0.07, crotoxin80 = 2.0 ± 0.05 ml g-1 min-1, P<0.05) and UF (control80 = 0.20 ± 0.02, venom80 = 0.32 ± 0.03, crotoxin80 = 0.70 ± 0.05 ml g-1 min-1, P<0.05), and decreased %TNa+ (control100 = 75.0 ± 2.3, venom100 = 62.9 ± 1.0, crotoxin80 = 69.0 ± 1.0 ml g-1 min-1, P<0.05). The infusion of crude venom tended to reduce PP, although the effect was not significant, whereas with crotoxin PP remained stable during the 100 min of perfusion. The kidneys perfused with crude venom and crotoxin showed abundant protein material in the urinary space and tubules. We conclude that Crotalus durissus terrificus venom and crotoxin, its major component, cause acute nephrotoxicity in the isolated rat kidney. The current experiments demonstrate a direct effect of venom and crotoxin on the perfused isolated kidney.