Endoplasmic reticulum stress inhibition ameliorated WFS1 expression alterations and reduced pancreatic islets' insulin secretion induced by high-fat diet in rats.

Endoplasmic reticulum (ER) stress is involved in the development of glucose homeostasis impairment. When ER stress occurs, the unfolded protein response (UPR) is activated to cope with it. One of the UPR components is WFS1 (Wolfram syndrome 1), which plays important roles in ER homeostasis and pancreatic islets glucose-stimulated insulin secretion (GSIS). Accordingly and considering that feeding high-fat food has a major contribution in metabolic disorders, this study aimed to investigate the possible involvement of pancreatic ER stress in glucose metabolism impairment induced by feeding high-fat diet (HFD) in male rats. After weaning, the rats were divided into six groups, and fed on normal diet and HFD for 20 weeks, then 4-phenyl butyric acid (4-PBA, an ER stress inhibitor) was administered. Subsequently, in all groups, after performing glucose tolerance test, the animals were dissected and their pancreases were removed to extract ER, islets isolation and assessment of GSIS. Moreover, the pancreatic ER stress [binding of immunoglobulin protein (BIP) and enhancer-binding protein homologous protein (CHOP)] and oxidative stress [malondialdehyde (MDA), glutathione (GSH) and catalase] biomarkers as well as WFS1 expression level were evaluated. HFD decreased pancreatic WFS1 protein and GSH levels, and enhanced pancreatic catalase activity, MDA content, BIP and CHOP protein and mRNA levels as well as Wfs1 mRNA amount. Accordingly, it increased BIP, CHOP and WFS1 protein levels in the extracted ER of pancreas. In addition, the HFD caused glucose intolerance, and decreased the islets' GSIS and insulin content. However, 4-PBA administration restored the alterations. It seems that, HFD consumption through inducing pancreatic ER stress, altered WFS1 expression levels, reduced the islets' GSIS and insulin content and finally impaired glucose homeostasis.

The Effect of 40-Hz White LED Therapy on Structure-Function of Brain Mitochondrial ATP-Sensitive Ca-Activated Large-Conductance Potassium Channel in Amyloid Beta Toxicity.

Photobiomodulation therapy has become the focus of medical research in many areas such as Alzheimer's disease (AD), because of its modulatory effect on cellular processes through light energy absorption via photoreceptors/chromophores located in the mitochondria. However, there are still many questions around the underlying mechanisms. This study was carried out to unravel whether the function-structure of ATP-sensitive mitoBKCa channels, as crucial components for maintenance of mitochondrial homeostasis, can be altered subsequent to light therapy in AD. Induction of Aß neurotoxicity in male Wistar rats was done by intracerebroventricular injection of Aß1-42. After a week, light-treated rats were exposed to 40-Hz white light LEDs, 15 min for 7 days. Electrophysiological properties of mitoBKCa channel were investigated using a channel incorporated into the bilayer lipid membrane, and mitoBKCa-ß2 subunit expression was determined using western blot analysis in Aß-induced toxicity and light-treated rats. Our results describe that conductance and open probability (Po) of mitoBKCa channel decreased significantly and was accompanied by a Po curve rightward shift in mitochondrial preparation in Aß-induced toxicity rats. We also showed a significant reduction in expression of mitoBKCa-ß2 subunit, which is partly responsible for a leftward shift in BKCa Po curve in low calcium status. Interestingly, we provided evidence of a significant improvement in channel conductance and Po after light therapy. We also found that light therapy improved mitoBKCa-ß2 subunit expression, increasing it close to saline group. The current study explains a light therapy improvement in brain mitoBKCa channel function in the Aß-induced neurotoxicity rat model, an effect that can be linked to increased expression of ß2 subunit.

Maternal stress induced endoplasmic reticulum stress and impaired pancreatic islets' insulin secretion via glucocorticoid receptor upregulation in adult male rat offspring.

Exposure to perinatal (prenatal and/or postnatal) stress is considered as a risk factor for metabolic disorders in later life. Accordingly, this study aimed to investigate the perinatal stress effects on the pancreatic endoplasmic reticulum (ER) stress induction, insulin secretion impairment and WFS1 (wolframin ER transmembrane Glycoprotein, which is involved in ER homeostasis and insulin secretion) expression changes, in rat offspring. According to the dams' period of exposure to variable stress, their male offspring were divided into, control (CTRL); pre-pregnancy, pregnancy, lactation stress (PPPLS); pre-pregnancy stress (PPS); pregnancy stress (PS); lactation stress (LS); pre-pregnancy, pregnancy stress (PPPS); pregnancy, lactation stress (PLS); pre-pregnancy, lactation stress (PPLS) groups. Offspring pancreases were removed for ER extraction and the assessment of ER stress biomarkers, WFS1 gene DNA methylation, and isolated islets' insulin secretion. Glucose tolerance was also tested. In the stressed groups, maternal stress significantly increased plasma corticosterone levels. In PPS, PS, and PPPS groups, maternal stress increased Bip (Hsp70; heat shock protein family A member 4), Chop (Ddit3; DNA- damage inducible transcript3), and WFS1 protein levels in pancreatic extracted ER. Moreover, the islets' insulin secretion and content along with glucose tolerance were impaired in these groups. In PPS, PS, LS and PPPS groups, the pancreatic glucocorticoid receptor (GR) expression increased. Maternal stress did not affect pancreatic WFS1 DNA methylation. Thus, maternal stress, during prenatal period, impaired the islets' insulin secretion and glucose homeostasis in adult male offspring, possibly through the induction of ER stress and GR expression in the pancreas, in this regard the role of WFS1 protein alteration in pancreatic ER should also be considered.

The 40-Hz White Light-Emitting Diode (LED) Improves the Structure-Function of the Brain Mitochondrial KATP Channel and Respiratory Chain Activities in Amyloid Beta Toxicity.

It has been described that using noninvasive exposure to 40-Hz white light LED reduces amyloid-beta, a peptide thought to initiate neurotoxic events in Alzheimer's disease (AD). However, the mechanisms remain to be identified. Since AD impairs mitochondrial potassium channels and respiratory chain activity, the objectives of the current study were to determine the effect of 40-Hz white light LED on structure-function of mitoKATP channel and brain mitochondrial respiratory chain activity, production of reactive oxygen species (ROS), and ΔΨm in AD. Single mitoKATP channel was considered using a channel incorporated into the bilayer lipid membrane and expression of mitoKATP-Kir6.1 subunit as a pore-forming subunit of the channel was determined using a western blot analysis in Aß1-42 toxicity and light-treated rats. Our results indicated a severe decrease in mito-KATP channel permeation and Kir6.1 subunit expression coming from the Aß1-42-induced neurotoxicity. Furthermore, we found that Aß1-42-induced neurotoxicity decreased activities of complexes I and IV and increased ROS production and ΔΨm. Surprisingly, light therapy increased channel permeation and mitoKATP-Kir6.1 subunit expression. Noninvasive 40-Hz white light LED treatment also increased activities of complexes I and IV and decreased ROS production and ΔΨm up to ~ 70%. Here, we report that brain mito-KATP channel and respiratory chain are, at least in part, novel targets of 40-Hz white light LED therapy in AD.

Intranasal insulin improves the structure-function of the brain mitochondrial ATP-sensitive Ca2+ activated potassium channel and respiratory chain activities under diabetic conditions.

Although it is well established that diabetes impairs mitochondrial respiratory chain activity, little is known of the effects of intranasal insulin (INI) on the mitochondrial respiratory chain and structure-function of mitoBKCa channel in diabetes. We have investigated this mechanism in an STZ-induced early type 2 diabetic model. Single ATP-sensitive mitoBKCa channel activity was considered in diabetic and INI-treated rats using a channel incorporated into the bilayer lipid membrane. Because mitoBKCa channels have been involved in mitochondrial respiratory chain activity, a study was undertaken to investigate whether the NADH, complexes I and IV, mitochondrial ROS production, and ΔΨm are altered in an early diabetic model. In this work, we provide evidence for a significant decrease in channel open probability and conductance in diabetic rats. Evidence has been shown that BKCa channel ß2 subunits induce a left shift in the BKCa channel voltage dependent curve in low Ca2+ conditions,; our results indicated a significant decrease in mitoBKCa ß2 subunits using Western blot analysis. Importantly, INI treatment improved mitoBKCa channel behaviors and ß2 subunits expression up to ~70%. We found that early diabetes decreased activities of complex I and IV and increased NADH, ROS production, and ΔΨm. Surprisingly, INI modified the mitochondrial respiratory chain, ROS production, and ΔΨm up to ~70%. Our results thus demonstrate an INI improvement in respiratory chain activity and ROS production in brain mitochondrial preparations coming from the STZ early diabetic rat model, an effect potentially linked to INI improvement in mitoBKCa channel activity and channel ß2 subunit expression.

A new brain mitochondrial sodium-sensitive potassium channel: effect of sodium ions on respiratory chain activity.

We have determined the electropharmacological properties of a new potassium channel from brain mitochondrial membrane using a planar lipid bilayer method. Our results show the presence of a channel with a conductance of 150 pS at potentials between 0 and -60 mV in 200 mM cis/50 mM trans KCl solutions. The channel was voltage independent, with an open probability value of approximately 0.6 at different voltages. ATP did not affect current amplitude or open probability at positive and negative voltages. Notably, adding iberiotoxin, charybdotoxin, lidocaine or margatoxin had no effect on the channel behavior. Similarly, no changes were observed by decreasing the cis pH to 6. Interestingly, the channel was inhibited by adding sodium in a dose-dependent manner. Our results also indicated a significant increase in mitochondrial complex IV activity and membrane potential and a decrease in complex I activity and mitochondrial ROS production in the presence of sodium ions. We propose that inhibition of mitochondrial potassium transport by sodium ions on potassium channel opening could be important for cell protection and ATP synthesis.

Paraventricular nucleus-microinjected glucose increases food intake in 18 h food-deprived rats: A central regulatory mechanism on serum ghrelin and leptin levels.

Our previous study demonstrated that glucose acts in hypothalamic paraventricular nucleus (PVN) to increase gastric acid secretion. However, there is no evidence to show the role of the hypothalamic PVN-microinjected glucose on food intake. On the other hand, it is known that ghrelin and leptin play important roles in food intake. The current study investigated the association between PVN-microinjected glucose and food intake and plasma ghrelin-leptin levels. After the PVN microinjection of glucose, food intake was measured. In other groups, ELIZA kits were used to obtain ghrelin, leptin, insulin and glucose concentrations. All experiments were done in18 h food-deprived rats. The findings revealed that the PVN-microinjected glucose increased food intake in a dose-dependent manner. The stimulatory effect of glucose persisted for more than 2 h. Interestingly, it was found that PVN microinjection of glucose stimulates plasma ghrelin and decreases plasma leptin levels without any effect on plasma insulin and glucose concentrations over 1 h. The results of the present study suggest that the PVN glucose-mediated cells may be involved in the regulatory mechanisms of food intake. This stimulatory effect seems to be mediated, at least in part, through central nervous system regulatory mechanisms of plasma leptin and ghrelin levels.

Ventral Tegmental Area Microinjected-SKF38393 Increases Regular Chow Intake in 18 Hours Food-Deprived Rats.

INTRODUCTION: Ventral Tegmental Area (VTA) dopamine neurons play an important role in reward mechanisms of food intake, and VTA dopamine receptors exist on the terminal of glutamatergic and GABAergic neurons and regulate Gamma-Aminobutyric Acid (GABA) and glutamate release. To our knowledge, no evidence indicates any role for VTA D1 dopamine receptors in regular chow intake. METHODS: In this paper, different dose of SKF38393, a D1 receptor agonist, was microinjected in VTA of 18-h food deprived-conscious rats and food intake was measured. RESULTS: Our results revealed that VTAmicroinjected SKF383993 increased regular chow intake in a dose-dependent manner. The SKF3833 stimulatory effect persisted over 2 h post-injection. The results showed that the SKF38393, at doses less than 5 µg, did not affect locomotor activities. CONCLUSION: VTA D1-like and/or serotonergic receptors may be involved in regulatory pathways. the current study suggests that VTA D1-like and/or serotonergic receptors not only affects food reward but is also involved in regulatory mechanisms of regular feeding.

Comparison of Effects of Light Anesthetics, Diethyl Ether and Carbon Dioxide, on Hypothalamic Paraventricular Nucleus D1 and D2 Dopamine Receptors- and Glucosensitive Neurons-Induced Food Intake in Fasted Conscious Rats.

INTRODUCTION: Carbon Dioxide (CO2) and diethyl ether are used as light anesthetics. However, experimental data about their side effects are scarce. In addition, in all our previous works on regulatory mechanisms of hypothalamus during food intake, including the effect of Paraventricular Nucleus (PVN) D1 and D2 dopamine receptors and glucosensitive neurons, the drug injections were performed under brief diethyl ether anesthesia. In the current study, we tested the hypothesis which postulates that CO2 and diethyl ether as light anesthetic agents affect the stimulatory effect of PVN dopamine receptors and glucosensitive neurons in feeding behavior. METHODS: Male Wistar rats were implanted with guide cannula directed to their PVN. Glucose (0.8 µg), SKF38393 (D1 agonist, 0.5 µg), quinpirole (D2 agonist, 0.3 µg) and saline (0.3 µL) were microinjected into the PVN and food intake was measured over 1 hour. RESULTS: Our results showed that CO2 but not diethyl ether decreased food intake compared to intact animals. The PVN injections of glucose, SKF38393, and quinpirole increased food intake under brief diethyl ether anesthesia. In contrast, the PVN microinjected glucose-induced and dopamine receptor agonists-induced food intake were inhibited under light CO2 anesthesia. CONCLUSION: Our results suggest that brief exposure to CO2 and diethyl ether as light anesthetic agents may affect PVN glucosensing neurons-induced and dopamine receptors-induced food intake in fasted rats.

Role of paraventricular hypothalamic dopaminergic D1 receptors in food intake regulation of food-deprived rats.

Dopaminergic neurons play an important role on central regulatory mechanisms of feeding behavior. Dopamine receptors are distributed within the hypothalamus and densely localized in the paraventricular hypothalamic nucleus (PVN). From these ideas we postulated that PVN D1 receptors may play a role in regulating the food intake behavioral process. In this paper, we considered the effects of SKF38393, a D1 receptor agonist, and the D1 receptor antagonist (SCH23390), on food intake of conscious rats deprived of food for 24h. Our findings revealed that intraparaventricular injections of SKF383993 (0.3-5µg) stimulated food intake behavior in a dose dependent manner. This stimulatory effect of SKF3833 persisted over 2h of the monitoring period. The PVN injections of D1 receptor antagonist were associated with dose-dependent inhibition of food intake. SCH23390 (0.01µg) was also administered 5min before intraparaventricular injection of SKF3833. The results showed that SCH23390 suppressed stimulated food intake induced by SKF38393 (1.2µg). In conclusion, endogenous dopamine impact PVN D1 receptors and may be a factor in regulating the food intake behavioral process.

Electro-pharmacological profiles of two brain mitoplast anion channels: Inferences from single channel recording.

We have characterized the conduction and blocking properties of two different chloride channels from brain mitochondrial inner membranes after incorporation into planar lipid bilayers. Our experiments revealed the existence of channels with a mean conductance of 158 ± 7 and 301 ± 8 pS in asymmetrical 200 mM cis/50 mM trans KCl solutions. We determined that the channels were ten times more permeable for Cl- than for K+, calculated from the reversal potential using the Goldman-Hodgkin-Katz equation. The channels were bell-shaped voltage dependent, with maximum open probability 0.9 at ± 20 mV. Two mitochondrial chloride channels were blocked after the addition of 10 µM DIDS. In addition, 158 pS chloride channel was blocked by 300 nM NPPB, acidic pH and 2.5 mM ATP, whereas the 301 pS chloride channel was blocked by 600 µM NPPB but not by acidic pH or ATP. Gating and conducting behaviors of these channels were unaffected by Ca2+. These results demonstrate that the 158 pS anion channel present in brain mitochondrial inner membrane, is probably identical to IMAC and 301 pS Cl channel displays different properties than those classically described for mitochondrial anion channels.

Evidence for a KATP Channel in Rough Endoplasmic Reticulum (rerKATP Channel) of Rat Hepatocytes.

We report in a previous study the presence of a large conductance K+ channel in the membrane of rough endoplasmic reticulum (RER) from rat hepatocytes incorporated into lipid bilayers. Channel activity in this case was found to decrease in presence of ATP 100 µM on the cytoplasmic side and was totally inhibited at ATP concentrations greater than 0.25 mM. Although such features would be compatible with the presence of a KATP channel in the RER, recent data obtained from a brain mitochondrial inner membrane preparation have provided evidence for a Maxi-K channel which could also be blocked by ATP within the mM concentration range. A series of channel incorporation experiments was thus undertaken to determine if the ATP-sensitive channel originally observed in the RER corresponds to KATP channel. Our results indicate that the gating and permeation properties of this channel are unaffected by the addition of 800 nM charybdotoxin and 1 µM iberiotoxin, but appeared sensitive to 10 mM TEA and 2.5 mM ATP. Furthermore, adding 100 µM glibenclamide at positive potentials and 400 µM tolbutamide at negative or positive voltages caused a strong inhibition of channel activity. Finally Western blot analyses provided evidence for Kir6.2, SUR1 and/or SUR2B, and SUR2A expression in our RER fractions. It was concluded on the basis of these observations that the channel previously characterized in RER membranes corresponds to KATP, suggesting that opening of this channel may enhance Ca2+ releases, alter the dynamics of the Ca2+ transient and prevent accumulation of Ca2+ in the ER during Ca2+ overload.

Gating behavior of endoplasmic reticulum potassium channels of rat hepatocytes in diabetes.

BACKGROUND: Defects in endoplasmic reticulum homeostasis are common occurrences in different diseases, such as diabetes, in which the function of endoplasmic reticulum is disrupted. It is now well established that ion channels of endoplasmic reticulum membrane have a critical role in endoplasmic reticulum luminal homeostasis. Our previous studies showed the presence of an ATP-sensitive cationic channel in endoplasmic reticulum. Therefore, in this study, we examined and compared the activities of this channel in control and diabetic rats using single-channel recording techniques. METHOD: Male Wistar rats were made diabetic for 2 weeks with a single dose injection of streptozotocin (45 mg/kg). Ion channel incorporation of rough endoplasmic reticulum of diabetic hepatocytes into the bilayer lipid membrane allowed the characterization of K+ channel. RESULTS: Ion channel incorporation of rough endoplasmic reticulum vesicles into the bilayer lipid revealed that the channel current-voltage (I-V) relation with a mean slope conductance of 520 ± 19 pS was unaffected in diabetes. Interestingly, the channel Po-voltage relation was significantly lower in diabetic rats at voltages above +30 mV. CONCLUSION: We concluded that the endoplasmic reticulum cationic channel is involved in diabetes. Also, this finding could be considered as a goal for further therapeutic plans.

Endoplasmic reticulum membrane potassium channel dysfunction in high fat diet induced stress in rat hepatocytes.

In a previous study we reported the presence of a large conductance K(+) channel in the membrane of endoplasmic reticulum (ER) from rat hepatocytes. The channel open probability (Po) appeared voltage dependent and reached to a minimum 0.2 at +50 mV. Channel activity in this case was found to be totally inhibited at ATP concentration 2.5 mM, glibenclamide 100 µM and tolbutamide 400 µM. Existing evidence indicates an impairment of endoplasmic reticulum functions in ER stress condition. Because ER potassium channels have been involved in several ER functions including cytoprotection, apoptosis and calcium homeostasis, a study was carried out to consider whether the ER potassium channel function is altered in a high fat diet model of ER stress. Male Wistar rats were made ER stress for 2 weeks with a high fat diet. Ion channel incorporation of ER stress model into the bilayer lipid membrane allowed the characterization of K(+) channel. Our results indicate that the channel Po was significantly increased at voltages above +30 mV. Interestingly, addition of ATP 7.5 mM, glibenclamide 400 µM and tolbutamide 2400 µM totally inhibited the channel activities, 3-fold, 4-fold and 6-fold higher than that in the control groups, respectively. Our results thus demonstrate a modification in the ER K(+) channel gating properties and decreased sensitivity to drugs in membrane preparations coming from ER high fat model of ER stress, an effect potentially linked to a change in ER K(+) channel subunits in ER stress condition. Our results may provide new insights into the cellular mechanisms underlying ER dysfunctions in ER stress.

Peripheral dopamine D2-like receptors have a regulatory effect on carbachol-, histamine- and pentagastrin-stimulated gastric acid secretion.

It has been documented that dopamine, an important regulator of gastric function in the brain-gut axis, has an inhibitory effect on the gastric acid secretion. It has also been suggested that dopamine D1, D2 and D5 receptor proteins are present in the gastrointestinal tract from the stomach through to the distal colon. Therefore, we hypothesized that peripheral D2 receptors may be involved in the control of stimulated gastric acid secretion. To address this question, we examined the effect of quinpirole, a selective D2 receptor-like agonist, and domperidone, a peripheral D2 receptor antagonist, on rat gastric acid secretion. Quinpirole (0.0001-0.5 mg/kg, i.p.) was administered simultaneously with intravenous infusions of histamine, pentagastrin, and carbachol. In some experiments, domperidone (3 and 7 mg/kg) was administered 30 min before quinpirole injection. We found that intraperitoneal injection of quinpirole (0.0001-0.5 mg/kg) suppressed stimulated gastric acid secretion induced by histamine (0.08 mg/100 g per h), pentagastrin (1 microg/100 g per h) and carbachol (4 microg/100 g per h) in a dose-dependent manner. This inhibitory effect of quinpirole persisted until the end of the experiments (90-120 min) and was completely suppressed by domperidone (7 mg/kg). In conclusion, the results of the present study suggest that peripheral D2-like receptors have an inhibitory effect on histaminergic-, pentagastrin- and cholinergic-stimulated gastric acid secretion. This inhibitory effect may be mediated by enteric dopaminergic neurons and/or non-neuronal membranes.

ATP regulation of a large conductance voltage-gated cation channel in rough endoplasmic reticulum of rat hepatocytes.

ATP-sensitive K+ channels play an important role in regulating membrane potential during metabolic stress. In this work we report the effect of ATP and ADP-Mg on a K+ channel present in the membrane of rough endoplasmic reticulum (RER) from rat hepatocytes incorporated into lipid bilayers. Channel activity was found to decrease in presence of ATP 100 microM on the cytoplasmic side and was totally inhibited at ATP concentrations greater than 0.25mM. The effect appeared voltage dependent, suggesting that the ATP binding site was becoming available upon channel opening. Channel activity was suppressed by the nonhydrolyzable ATP analog (ATPgammaS), ruling out a phosphorylation-based mechanism. Notably addition of 2.5mM ADP-Mg to the cytosolic side increased the channel open probability at negative potentials. We conclude that the large conductance voltage-gated cation channel in RER of rat hepatocytes is an ATP and ADP sensitive channel likely to be involved in cellular processes such as Ca(2+) signaling or control of membrane potential across the endoplasmic reticulum membrane.

Evidence for a large conductance voltage gated cationic channel in rough endoplasmic reticulum of rat hepatocytes.

In this work, we report the single channel characterization of a voltage gated cationic channel from rough endoplasmic reticulum (RER) membranes of rat hepatocytes incorporated into a planar lipid bilayer. The channel was found to be cation selective with a main conductance of 598+/-20 pS in 200 mM KCl cis/50 mM KCl trans. The channel open probability appeared voltage dependent with a voltage for half activation (V(1/2)) of 38 mV and an effective gating charge z of -6.66. Adding either 4-AP (5 mM) or ATP (2.5 mM) to the side corresponding to the cell internal medium caused a strong inhibition of the channel activity. This channel is likely to be involved in maintaining proper cation homeostasis in the RER of hepatocytes.