Decreasing lactate level and increasing antibody production in Chinese Hamster Ovary cells (CHO) by reducing the expression of lactate dehydrogenase and pyruvate dehydrogenase kinases

Large-scale fed-batch cell culture processes of CHO cells are the standard platform for the clinical and commercial production of monoclonal antibodies. Lactate is one of the major by-products of CHO fed-batch culture. In pH-controlled bioreactors, accumulation of high levels of lactate is accompanied by high osmolality due to the addition of base to control pH of the cell culture medium, potentially leading to lower cell growth and lower therapeutic protein production during manufacturing. Lactate dehydrogenase (LDH) is an enzyme that catalyzes the conversion of the substrate, pyruvate, into lactate and many factors including pyruvate concentration modulate LDH activity. Alternately, pyruvate can be converted to acetyl-CoA by pyruvate dehydrogenases (PDHs), to be metabolized in the TCA cycle. PDH activity is inhibited when phosphorylated by pyruvate dehydrogenase kinases (PDHKs). In this study, we knocked down the gene expression of lactate dehydrogenase A (LDHa) and PDHKs to investigate the effect on lactate metabolism and protein production. We found that LDHa and PDHKs can be successfully downregulated simultaneously using a single targeting vector carrying small inhibitory RNAs (siRNA) for LDHa and PDHKs. Moreover, our fed-batch shake flask evaluation data using siRNA-mediated LDHa/PDHKs knockdown clones showed that downregulating LDHa and PDHKs in CHO cells expressing a therapeutic monoclonal antibody reduced lactate production, increased specific productivity and volumetric antibody production by approximately 90%, 75% and 68%, respectively, without appreciable impact on cell growth. Similar trends of lower lactate level and higher antibody productivity on average in siRNA clones were also observed from evaluations performed in bioreactors.     

Cannabinoid Type 1 and Type 2 Receptor Antagonists Prevent Attenuation of Serotonin-Induced Reflex Apneas by Dronabinol in Sprague-Dawley Rats

The prevalence of obstructive sleep apnea (OSA) in Americans is 9% and increasing. Increased afferent vagal activation may predispose to OSA by reducing upper airway muscle activation/patency and disrupting respiratory rhythmogenesis. Vagal afferent neurons are inhibited by cannabinoid type 1 (CB₁) or cannabinoid type 2 (CB₂) receptors in animal models of vagally-mediated behaviors. Injections of dronabinol, a non-selective CB₁/CB₂ receptor agonist, into the nodose ganglia reduced serotonin (5-HT)-induced reflex apneas. It is unknown what role CB₁ and/or CB₂ receptors play in reflex apnea. Here, to determine the independent and combined effects of activating CB₁ and/or CB₂ receptors on dronabinol’s attenuating effect, rats were pre-treated with CB₁ (AM251) and/or CB₂ (AM630) receptor antagonists. Adult male Sprague-Dawley rats were anesthetized, instrumented with bilateral electrodes to monitor genioglossus electromyogram (EMGgg) and a piezoelectric strain gauge to monitor respiratory pattern. Following intraperitoneal treatment with AM251 and/or AM630, or with vehicle, serotonin was intravenously infused into a femoral vein to induce reflex apnea. After baseline recordings, the nodose ganglia were exposed and 5-HT-induced reflex apneas were again recorded to confirm that the nerves remained functionally intact. Dronabinol was injected into each nodose ganglion and 5-HT infusion was repeated. Prior to dronabinol injection, there were no significant differences in 5-HT-induced reflex apneas or phasic and tonic EMGgg before or after surgery in the CB₁, CB₂, combined CB₁/CB₂ antagonist, and vehicle groups. In the vehicle group, dronabinol injections reduced 5-HT-induced reflex apnea duration. In contrast, dronabinol injections into nodose ganglia of the CB₁, CB₂, and combined CB₁/CB₂ groups did not attenuate 5-HT-induced reflex apnea duration. However, the CB₁ and CB₂ antagonists had no effect on dronabinol’s ability to increase phasic EMGgg. These findings underscore the therapeutic potential of dronabinol in the treatment of OSA and implicate participation of both cannabinoid receptors in dronabinol’s apnea suppression effect.     

CB1 receptor signaling regulates social anxiety and memory

The endocannabinoid (eCB) system regulates emotion, stress, memory and cognition through the cannabinoid type 1 (CB₁) receptor. To test the role of CB₁ signaling in social anxiety and memory, we utilized a genetic knockout (KO) and a pharmacological approach. Specifically, we assessed the effects of a constitutive KO of CB₁ receptors (CB₁KOs) and systemic administration of a CB₁ antagonist (AM251; 5 mg/kg) on social anxiety in a social investigation paradigm and social memory in a social discrimination test. Results showed that when compared with wild‐type (WT) and vehicle‐treated animals, CB₁KOs and WT animals that received an acute dose of AM251 displayed anxiety‐like behaviors toward a novel male conspecific. When compared with WT animals, KOs showed both active and passive defensive coping behaviors, i.e. elevated avoidance, freezing and risk‐assessment behaviors, all consistent with an anxiety‐like profile. Animals that received acute doses of AM251 also showed an anxiety‐like profile when compared with vehicle‐treated animals, yet did not show an active coping strategy, i.e. changes in risk‐assessment behaviors. In the social discrimination test, CB₁KOs and animals that received the CB₁ antagonist showed enhanced levels of social memory relative to their respective controls. These results clearly implicate CB₁ receptors in the regulation of social anxiety, memory and arousal. The elevated arousal/anxiety resulting from either total CB₁ deletion or an acute CB₁ blockade may promote enhanced social discrimination/memory. These findings may emphasize the role of the eCB system in anxiety and memory to affect social behavior .     

Cannabinoid receptor 1 mediates palmitic acid‐induced apoptosis via endoplasmic reticulum stress in human renal proximal tubular cells

The endocannabinoid system (ECS) is activated at the onset of obesity and diverse metabolic diseases. Endocannabinoids mediate their physiological and behavioral effects by activating specific cannabinoid receptors, mainly cannabinoid receptor 1 (CB₁R). Diabetic nephropathy (DN) is induced by hyperlipidemia, and renal proximal tubule cells are an important site for the onset of DN. However, the pathophysiology of CB₁R, especially in the hyperlipidemia of DN, has not been elucidated. Therefore, we examined the effect of palmitic acid (PA) on CB₁R expression and its related signal pathways in human renal proximal tubular cells (HK‐2 cells). PA significantly increased CB₁R mRNA and protein levels and induced CB₁R internalization. PA‐induced activation of CB₁R is prevented by the treatment of AACOCF₃ (a cPLA₂ inhibitor), indomethacin and NS398 (a COX 2 inhibitors). Indeed, PA increased cPLA₂, and COX‐2 but not COX‐1. We also investigated whether the PA‐induced activation of CB₁R is linked to apoptosis. As a result, AM251 (a CB₁R antagonist) attenuated PA‐mediated apoptosis in a concentration‐dependent manner. Furthermore, PA decreased GRP78 expression and induced increases in the endoplasmic reticulum (ER) stress signaling pathways p‐PERK, p‐eIF2α, p‐ATF4, and CHOP, which were blocked by AM251 treatment. Moreover, PA increased the Bax/Bcl‐2 ratio, cleaved PARP, and caspase‐3 levels. The PA‐induced apoptotic effects were decreased with CB₁R‐specific antagonist (AM251) treatment and CB1 si‐RNA transfection. In conclusion, PA induced apoptosis through ER stress via CB₁R expression in human proximal tubule cells. Our results provide evidence that CB₁R blockade may be a potential anti‐diabetic therapy for the treatment of DN. J. Cell. Physiol. 225: 654–663, 2010. © 2010 Wiley‐Liss, Inc.     

THC Prevents MDMA Neurotoxicity in Mice

The majority of MDMA (ecstasy) recreational users also consume cannabis. Despite the rewarding effects that both drugs have, they induce several opposite pharmacological responses. MDMA causes hyperthermia, oxidative stress and neuronal damage, especially at warm ambient temperature. However, THC, the main psychoactive compound of cannabis, produces hypothermic, anti-inflammatory and antioxidant effects. Therefore, THC may have a neuroprotective effect against MDMA-induced neurotoxicity. Mice receiving a neurotoxic regimen of MDMA (20 mg/kg ×4) were pretreated with THC (3 mg/kg ×4) at room (21°C) and at warm (26°C) temperature, and body temperature, striatal glial activation and DA terminal loss were assessed. To find out the mechanisms by which THC may prevent MDMA hyperthermia and neurotoxicity, the same procedure was carried out in animals pretreated with the CB₁ receptor antagonist AM251 and the CB₂ receptor antagonist AM630, as well as in CB₁, CB₂ and CB₁/CB₂ deficient mice. THC prevented MDMA-induced-hyperthermia and glial activation in animals housed at both room and warm temperature. Surprisingly, MDMA-induced DA terminal loss was only observed in animals housed at warm but not at room temperature, and this neurotoxic effect was reversed by THC administration. However, THC did not prevent MDMA-induced hyperthermia, glial activation, and DA terminal loss in animals treated with the CB₁ receptor antagonist AM251, neither in CB₁ and CB₁/CB₂ knockout mice. On the other hand, THC prevented MDMA-induced hyperthermia and DA terminal loss, but only partially suppressed glial activation in animals treated with the CB₂ cannabinoid antagonist and in CB₂ knockout animals. Our results indicate that THC protects against MDMA neurotoxicity, and suggest that these neuroprotective actions are primarily mediated by the reduction of hyperthermia through the activation of CB₁ receptor, although CB₂ receptors may also contribute to attenuate neuroinflammation in this process.     

Contribution of hypothermia and CB1 receptor activation to protective effects of TAK-937, a cannabinoid receptor agonist, in rat transient MCAO model

Background

Cannabinoid (CB) receptor agonists are expected to alleviate ischemic brain damage by modulating neurotransmission and neuroinflammatory responses via CB₁ and CB₂ receptors, respectively. In a previous study, TAK-937, a novel potent and selective CB₁ and CB₂ receptor agonist, was shown to exert significant cerebroprotective effects accompanied by hypothermia after transient middle cerebral artery occlusion (MCAO) in rats. Sustained hypothermia itself induces significant neuroprotective effects. In the present studies, we examined the relative contribution of hypothermia and CB₁ receptor activation to the cerebroprotective effects of TAK-937.

Methodology/Principal Findings

Using a multichannel brain temperature controlling system we developed, the brain temperature of freely moving rats was telemetrically monitored and maintained between 37 and 38°C during intravenous infusion of TAK-937 (100 µg/kg/h) or vehicle for 24 h after 2 h MCAO. AM251, a selective CB₁ receptor antagonist, was administered intraperitoneally at 30 mg/kg 30 min before starting intravenous infusion of TAK-937 (100 µg/kg/h) for 24 h. Rats were sacrificed and their brains were isolated 26 h after MCAO in both experiments. When the hypothermic effect of TAK-937 was completely reversed by a brain temperature controlling system, the infarct-reducing effect of TAK-937 was attenuated in part, but remained significant. On the other hand, concomitant AM251 treatment with TAK-937 completely abolished the hypothermic and infarct-reducing effects of TAK-937.

Conclusions/Significance

We conclude that the cerebroprotective effects of TAK-937 were at least in part mediated by induction of hypothermia, and mainly mediated by CB₁ receptor activation.     

Expression and Functional Relevance of Cannabinoid Receptor 1 in Hodgkin Lymphoma

Background

Cannabinoid receptor 1 (CB₁) is expressed in certain types of malignancies. An analysis of CB₁ expression and function in Hodgkin lymphoma (HL), one of the most frequent lymphomas, was not performed to date.

Design and Methods

We examined the distribution of CB₁ protein in primary cases of HL. Using lymphoma derived cell lines, the role of CB₁ signaling on cell survival was investigated.

Results

A predominant expression of CB₁ was found in Hodgkin-Reed-Sternberg cells in a vast majority of classical HL cases. The HL cell lines L428, L540 and KM-H2 showed strong CB₁-abundance and displayed a dose-dependent decline of viability under CB₁ inhibition with AM251. Further, application of AM251 led to decrease of constitutively active NFκB/p65, a crucial survival factor of HRS-cells, and was followed by elevation of apoptotic markers in HL cells.

Conclusions

The present study identifies CB₁ as a feature of HL, which might serve as a potential selective target in the treatment of Hodgkin lymphoma.     

Effects of progesterone on glucose metabolism in isolated acini from mammary glands of lactating rats

Isolated acini from lactating rat mammary gland were incubated with glucose (5 mm) and progesterone. The steroid (0.1 mm) decreased glucose utilization and pyruvate accumulation, but increased the formation of lactate. The production of ¹⁴CO₂ and ¹⁴C-labeled lipid from [1-¹⁴C]glucose, and the incorporation of ³H₂O into lipid were also inhibited by progesterone. At lower concentrations of progesterone (0.01–0.025 mm) the only effects were an increased [lactate], a decreased [pyruvate], and a consequent rise in the lactate/pyruvate ratio. Addition of dichloroacetate, an activator of pyruvate dehydrogenase, did not reverse these effects and assays of active pyruvate dehydrogenase showed no inactivation by progesterone. The steroid did not affect pyruvate utilization but markedly inhibited the removal of lactate, suggesting that progesterone causes a decreased reoxidation of cytosolic NADH and thus alters the cytosolic redox state. The findings are discussed in relation to the physiological role of progesterone during pregnancy and lactation.     

Likely individuality of the enzymes catalyzing the phosphorylation of choline and ethanolamine.

Dichloroacetate has effects upon hepatic metabolism which are profoundly different from its effects on heart, skeletal muscle, and adipose tissue metabolism. With hepatocytes prepared from meal-fed rats, dichloroacetate was found to activate pyruvate dehydrogenase, to increase the utilization of lactate and pyruvate without effecting an increase in the net utilization of glucose, to increase the rate of fatty acid synthesis, and to decrease slightly [1-¹⁴C]oleate oxidation to ¹⁴CO₂ without decreasing ketone body formation. With hepatocytes isolated from 48-h-starved rats, dichloroacetate was found to activate pyruvate dehydrogenase, to have no influence on net glucose utilization, to inhibit gluconeogenesis slightly with lactate as substrate, and to stimulate gluconeogenesis significantly with alanine as substrate. The stimulation of fatty acid synthesis by dichloroacetate suggests that the activity of pyruvate dehydrogenase can be rate determining for fatty acid synthesis in isolated liver cells. The minor effects of dichloroacetate on gluconeogenesis suggest that the regulation of pyruvate dehydrogenase is only of marginal importance in the control of gluconeogenesis.     

Effect of dichloroacetate and glucagon on the incorporation of labeled substrates into glucose and on pyruvate dehydrogenase in hepatocytes from fed and starved rats

Dichloroacetate (2 mm) stimulated the conversion of [1-¹⁴C]lactate to glucose in hepatocytes from fed rats. In hepatocytes from rats starved for 24 h, where the mitochondrial $\text{NADH}\text{NAD}{}^{\mathrm{+}}$ ratio is elevated, dichloroacetate inhibited the conversion of [1-¹⁴C]lactate to glucose. Dichloroacetate stimulated ¹⁴CO₂ production from [1-¹⁴C]lactate in both cases. It also completely activated pyruvate dehydrogenase and increased flux through the enzyme. The addition of β-hydroxybutyrate, which elevates the intramitochondrial $\text{NADH}\text{NAD}{}^{\mathrm{+}}$ ratio, changed the metabolism of [1-¹⁴C]lactate in hepatocytes from fed rats to a pattern similar to that seen in hepatocytes from starved rats. Thus, the effect of dichloroacetate on labeled glucose synthesis from lactate appears to depend on the mitochondrial oxidation-reduction state of the hepatocytes. Glucagon (10 nm) stimulated labeled glucose synthesis from lactate or alanine in hepatocytes from both fed and starved rats and in the absence or presence of dichloroacetate. The hormone had no effect on pyruvate dehydrogenase activity whether or not the enzyme had been activated by dichloroacetate. Thus, it appears that pyruvate dehydrogenase is not involved in the hormonal regulation of gluconeogenesis. Glucagon inhibited the incorporation of 10 mm [1-¹⁴C]pyruvate into glucose in hepatocytes from starved rats. This inhibition has been attributed to an inhibition of pyruvate dehydrogenase by the hormone (Zahlten et al., 1973, Proc. Nat. Acad. Sci. USA70, 3213–3218). However, dichloroacetate did not prevent the inhibition of glucose synthesis. Nor did glucagon alter the activity of pyruvate dehydrogenase in homogenates of cells that had been incubated with 10 mm pyruvate in the absence or presence of dichloroacetate. Thus, the inhibition by glucagon of pyruvate gluconeogenesis does not appear to be due to an inhibition of pyruvate dehydrogenase.     

Effectors of fatty acid synthesis in hepatoma tissue culture cells

An investigation was undertaken to better understand the process of fatty acid synthesis in hepatoma tissue culture (HTC) cells. By comparing the findings to the normal liver some of the differences between normal and cancer tissue were defined. Incubation of the HTC cells in a buffered salt-defatted albumin medium showed that fatty acid synthesis was dependent upon the addition of substrate. The order of stimulation was glucose + pyruvate ～- glucose + alanine ～- glucose + lactate ～- pyruvate > glucose > alanine ? no additions. Fatty acid synthesis in HTC cells was decreased by oleate. In these respects HTC cells are similar to the liver; however, in contrast to the normal liver, N⁶, O²-dibutyryl cyclic adenosine 3′,5′-monophosphate (dibutyryl-cAMP) did not inhibit glycolysis or fatty acid synthesis. The cytoplasmic redox potential, as reflected by the lactate to pyruvate ratio, was found to be elevated compared to normal liver but unchanged by the addition of dibutyryl cAMP. Since higher rates of fatty acid synthesis are associated with lower lactate-to-pyruvate ratios in normal liver, it was expected that by decreasing the lactate-to-pyruvate ratio in HTC cells the rate of fatty acid synthesis would increase. One way to lower the lactate to pyruvate ratio is to increase the activity of the malate-aspartate shuttle. Stimulators of the hepatic malate-aspartate shuttle in normal liver (ammonium ion, glutamine, and lysine) had mixed effects on the redox state and fatty acid synthesis in HTC cells. Both ammonium ion and glutamine decreased the redox potential and increased the rate of fatty acid synthesis. Lysine was without effect on either process. Since NH₄Cl and glutamine stimulate the movement of reducing equivalents into the mitochondria and decrease the redox potential, then the stimulation of fatty acid synthesis by NH₄Cl and glutamine may be due to an increase in the movement of reducing equivalents into the mitochondria. However, if the shuttle were rate determining for fatty acid synthesis the rate from added lactate would be the same as from glucose alone but would be lower than from pyruvate which does not require the movement of reducing equivalents. This was not the case. Lactate and pyruvate gave comparable rates which were higher than glucose alone. Other possible sites of stimulation were investigated. The possibility that NH₄⁺ and glutamine stimulated fatty acid synthesis by activating pyruvate dehydrogenase was excluded by finding that dichloroacetate, an activator of pyruvate dehydrogenase, did not stimulate fatty acid synthesis when glucose was added. Stimulation by NH₄⁺ and glutamine at steps beyond pyruvate dehydrogenase was ruled out by the observation that NH₄⁺ caused no stimulation from added pyruvate. NH₄⁺ and glutamine did not alter the pentose phosphate pathway as determined by ¹⁴CO₂ production from [1-¹⁴C]- or [6-¹⁴C]glucose. Ammonium ion and glutamine increased glucose consumption and increased lactate and pyruvate accumulation. The increased glycolysis in HTC cells appears to be the explanation for the stimulation of fatty acid synthesis by NH₄⁺ and glutamine, even though glycolysis is much more rapid than fatty acid synthesis in these cells. The following observations support this conclusion. First, the percentage increase in glycolysis caused by NH₄⁺ or glutamine is closely matched by the percentage increase in fatty acid synthesis. Second, the malate-aspartate shuttle, the pentose phosphate pathway, and the steps past pyruvate are not limiting in the absence of NH₄⁺ or glutamine.     

Interstitial lactate,lactate/pyruvate and glucose in rat muscle before,during and in the recovery from global hypoxia

Background

Hypoxia results in an imbalance between oxygen supply and oxygen consumption. This study utilized microdialysis to monitor changes in the energy-related metabolites lactate, pyruvate and glucose in rat muscle before, during and after 30 minutes of transient global hypoxia. Hypoxia was induced in anaesthetised rats by reducing inspired oxygen to 6% O₂ in nitrogen.

Results

Basal values for lactate, the lactate/pyruvate ratio and glucose were 0.72 ± 0.04 mmol/l, 10.03 ± 1.16 and 3.55 ± 0.19 mmol/l (n = 10), respectively. Significant increases in lactate and the lactate/pyruvate ratio were found in the muscle after the induction of hypoxia. Maximum values of 2.26 ± 0.37 mmol/l for lactate were reached during early reperfusion, while the lactate/pyruvate ratio reached maximum values of 35.84 ± 7.81 at the end of hypoxia. Following recovery to ventilation with air, extracellular lactate levels and the lactate/pyruvate ratio returned to control levels within 30-40 minutes. Extracellular glucose levels showed no significant difference between hypoxia and control experiments.

Conclusions

In our study, the complete post-hypoxic recovery of metabolite levels suggests that metabolic enzymes of the skeletal muscle and their related cellular components may be able to tolerate severe hypoxic periods without prolonged damage. The consumption of glucose in the muscle in relation to its delivery seems to be unaffected.

     

HIF-1-mediated expression of pyruvate dehydrogenase kinase: a metabolic switch required for cellular adaptation to hypoxia

Activation of glycolytic genes by HIF-1 is considered critical for metabolic adaptation to hypoxia through increased conversion of glucose to pyruvate and subsequently to lactate. We found that HIF-1 also actively suppresses metabolism through the tricarboxylic acid cycle (TCA) by directly trans-activating the gene encoding pyruvate dehydrogenase kinase 1 (PDK1). PDK1 inactivates the TCA cycle enzyme, pyruvate dehydrogenase (PDH), which converts pyruvate to acetyl-CoA. Forced PDK1 expression in hypoxic HIF-1alpha null cells increases ATP levels, attenuates hypoxic ROS generation, and rescues these cells from hypoxia-induced apoptosis. These studies reveal a hypoxia-induced metabolic switch that shunts glucose metabolites from the mitochondria to glycolysis to maintain ATP production and to prevent toxic ROS production.     

Engineering of Corynebacterium glutamicum as a prototrophic pyruvate-producing strain: Characterization of a ramA-deficient mutant and its application for metabolic engineering

To construct a prototrophic Corynebacterium glutamicum strain that efficiently produces pyruvate from glucose, the effects of inactivating RamA, a global regulator responsible for activating the oxidative tricarboxylic acid (TCA) cycle, on glucose metabolism were investigated. ΔramA showed an increased specific glucose consumption rate, decreased growth, comparable pyruvate production, higher formation of lactate and acetate, and lower accumulation of succinate and 2-oxoglutarate compared to the wild type. A significant decrease in pyruvate dehydrogenase complex activity was observed for ΔramA, indicating reduced carbon flow to the TCA cycle in ΔramA. To create an efficient pyruvate producer, the ramA gene was deleted in a strain lacking the genes involved in all known lactate- and acetate-producing pathways. The resulting mutant produced 161 mM pyruvate from 222 mM glucose, which was significantly higher than that of the parent (89.3 mM; 1.80-fold).     

Stimulation of flux through hepatic pyruvate dehydrogenase by 3-mercaptopicolinate

Isolated hepatocytes from 24-h-starved rats were used to assess the possible effect of Ahe hypoglycaemic agent 3-mercaptopicolinate on flux through the hepatic pyruvate dehydrogenase complex. Increasing the extraceIIular pyruvate concentration from 1 mM to 2 mM or 5 mM resulted in an increase in flux through pyruvate dehydrogenase and the tricarboxylic acid cycle as measured by¹⁴CO₂ evolution from [1-¹⁴C]pyruvate and [3-¹⁴C]pyruvate. Gluconeogenesis was inhibited by 3-mercaptopicolinate from both 1 mM and 2 mM pyruvate, but significant increases in malate and citrate concentrations only occurred in cells incubated with 1 mM pyruvate. Flux through pyruvate dehydrogenase was stimulated by 3-mercaptopicolinate with 1 mM pyruvate but was unaltered with 2 mM pyruvate. Dichloroacetate stimulated flux through pyruvate dehydrogenase with no effect on gluconeogenesis in the presence of I mM pyruvate. There was no effect of 3-mercaptopicolinate, administered in vivo, to 24-h-starved rats on the activity of pyruvate dehydrogenase in freeze-clamped heart or liver tissue, although the drug did decrease blood glucose concentration and increase the blood concentrations of lactate and alanine. Dichloroacetate, administered in vivo to 24-h-starved rats, increased the activity of pyruvate dehydrogenase in freeze-clamped heart and liver, and caused decreases in the blood concentrations of glucose, lactate , and alanine. The results suggest that 3-mercaptopicolinate increases flux through hepatocyte pyruvate dehydrogenase by an indirect mechanism.     

Metabolic effects of cannabinoids in zebrafish (<Emphasis Type="Italic">Danio rerio</Emphasis>) embryos determined by <Superscript>1</Superscript>H NMR metabolomics

The study of drug-driven biochemical changes is important in order to determine the biomarkers associated with a specific compound activity in an individual biological system. Rodent models have been widely used to study the metabolic changes induced by psychostimulants in a cell, tissue or whole organism. However these models are not suitable for large-scale, high-throughput screening. Here, we used zebrafish embryos to study the metabolic effects of the cannabinoid receptor type 1 (CB₁) agonist (?)-(6aR,10aR)-6,6,9-trimethyl-3-pentyl-6a,7,8,10a-tetrahydro-6H-benzo[c]chromen-1-ol (?⁹-THC) and antagonist (AM251). The zebrafish embryos were exposed to ?⁹-THC and AM251 at 24 h post fertilization (hpf) for 96 h. Proton nuclear magnetic resonance based metabolomic results show an increase in the level of choline, betaine, taurine, adenosine triphosphate and glucose upon exposure to ?⁹-THC. The levels of excitatory neurotransmitters (glutamate and glutamine) increased at lower doses of ?⁹-THC, whereas toxic dose resulted in reduction of glutamate. In contrast to ?⁹-THC, AM251 caused a dose dependent reduction of betaine, choline, taurine and also reduce the level of glutamate and glutamine. Interestingly, both compounds induce the production of the dopamine precursors, phenylalanine and tyrosine at higher doses. These findings suggest that CB₁ receptor is involved in the regulation of metabolites, which might be involved in the neurotransmission of zebrafish embryos. Furthermore, our results show that zebrafish embryo can be successfully used to provide a detailed overview of general effects of drug on the overall metabolome of an intact organism.     

CB<Subscript>1</Subscript> Receptors Mediated Inhibition of ATP-Induced [Ca<Superscript>2+</Superscript>]i Increase in Cultured Rat Spinal Dorsal Horn Neurons

Spinal cannabinoid receptor 1 (CB₁R) and purinergic P2X receptors (P2XR) play a critical role in the process of pathological pain. Both CB₁R and P2XR are expressed in spinal dorsal horn (DH) neurons. It is not clear whether CB₁ receptor activation modulates the function of P2X receptor channels within dorsal horn. For this reason, we observed the effect of CP55940 (cannabinoid receptor agonist) on ATP-induced Ca²⁺ mobilization in cultured rat DH neurons. The changes of intracellular calcium concentration ([Ca²⁺]i) were detected with confocal laser scanning microscopy using fluo-4/AM as a calcium fluorescent indicator. 100 μM ATP caused [Ca²⁺]i increase in cultured DH neurons. ATP-evoked [Ca²⁺]i increase in DH neurons was blocked by chelating extracellular Ca²⁺ and P2 purinoceptor antagonist PPADS. At the same time, ATP-γ-S (a non-hydrolyzable ATP analogue) mimicked the ATP action, while P2Y receptor agonist ADP failed to evoke [Ca²⁺]i increase in cultured DH neurons. These data suggest that ATP-induced [Ca²⁺]i elevation in cultured DH neurons is mediated by P2X receptor. Subsequently, we noticed that, in cultured rat DH neurons, ATP-induced Ca²⁺ mobilization was inhibited after pretreated with CP55940 with a concentration-dependent manner, which implies that the opening of P2X receptor channels are down-regulated by activation of cannabinoid receptor. The inhibitory effect of CP55940 on ATP-induced Ca²⁺ response was mimicked by ACEA (CB₁R agonist), but was not influenced by AM1241 (CB₂R agonist). Moreover, the inhibitory effect of CP55940 on ATP-induced Ca²⁺ mobilization was blocked by AM251 (CB₁ receptor antagonist), but was not influenced by AM630 (CB₂ receptor antagonist). In addition, we also observed that forskolin (an activator of adenylate cyclase) and 8-Br-cAMP (a cell-permeable cAMP analog) reversed the inhibitory effect of CP55940, respectively. In a summary, our observations raise a possibility that CB₁R rather than CB₂R can downregulate the opening of P2X receptor channels in DH neurons. The reduction of cAMP/PKA signaling is a key element in the inhibitory effect of CB₁R on P2X-channel-induced Ca²⁺ mobilization.     

Differential modulation of glucose,lactate, and pyruvate oxidation by insulin and dichloroacetate in the rat heart

Despite the fact that lactate and pyruvate are potential substrates for energy production in vivo, our understanding of the control and regulation of carbohydrate metabolism is based principally on studies where glucose is the only available carbohydrate. Therefore, the purpose of this study was to determine the contributions of lactate, pyruvate, and glucose to energy production in the isolated, perfused rat heart over a range of insulin concentrations and after activation of pyruvate dehydrogenase with dichloroacetate (DCA). Hearts were perfused with physiological concentrations of [1-13C]glucose, [U-13C]lactate, [2-13C]pyruvate, and unlabeled palmitate for 45 min. Hearts were freeze clamped, and 13C NMR glutamate isotopomer analysis was performed on tissue extracts. Glucose, lactate, and pyruvate all contributed significantly to myocardial energy production; however, in the absence of insulin, glucose contributed only 25-30% of total pyruvate oxidation. Even under conditions where carbohydrates represented >95% of substrate entering the tricarboxylic acid (TCA) cycle, we found that glucose contributed at most 50-60% of total carbohydrate oxidation. Despite being present at only 0.1 mM, pyruvate contributed between approximately 10% and 30% of total acetyl-CoA entry into the TCA cycle. We also found that insulin and DCA not only increased glucose oxidation but also exogenous pyruvate oxidation; however, lactate oxidation was not increased. The differential effects of insulin and DCA on pyruvate and lactate oxidation provide further evidence for compartmentation of cardiac carbohydrate metabolism. These results may have important implications for understanding the mechanisms underlying the beneficial effects of increasing cardiac carbohydrate metabolism.     

Evidence for a higher glycolytic than oxidative metabolic activity in white matter of rat brain

Different values exist for glucose metabolism in white matter; it appears higher when measured as accumulation of 2-deoxyglucose than when measured as formation of glutamate from isotopically labeled glucose, possibly because the two methods reflect glycolytic and tricarboxylic acid (TCA) cycle activities, respectively. We compared glycolytic and TCA cycle activity in rat white structures (corpus callosum, fimbria, and optic nerve) to activities in parietal cortex, which has a tight glycolytic-oxidative coupling. White structures had an uptake of [(3)H]2-deoxyglucose in vivo and activities of hexokinase, glucose-6-phosphate isomerase, and lactate dehydrogenase that were 40-50% of values in parietal cortex. In contrast, formation of aspartate from [U-(14)C]glucose in awake rats (which reflects the passage of (14)C through the whole TCA cycle) and activities of pyruvate dehydrogenase, citrate synthase, alpha-ketoglutarate dehydrogenase, and fumarase in white structures were 10-23% of cortical values, optic nerve showing the lowest values. The data suggest a higher glycolytic than oxidative metabolism in white matter, possibly leading to surplus formation of pyruvate or lactate. Phosphoglucomutase activity, which interconverts glucose-6-phosphate and glucose-1-phosphate, was similar in white structures and parietal cortex ( approximately 3 nmol/mg tissue/min), in spite of the lower glucose uptake in the former, suggesting that a larger fraction of glucose is converted into glucose-1-phosphate in white than in gray matter. However, the white matter glycogen synthase level was only 20-40% of that in cortex, suggesting that not all glucose-1-phosphate is destined for glycogen formation.     

D2 receptor-mediated inhibition of dopamine release in the rat striatum in vitro is modulated by CB1 receptors: studies using fast cyclic voltammetry

Cannabinoid CB₁ receptors are highly expressed in the striatum where they are known to be co‐localized with dopamine D₂ receptors. There is now strong evidence that cannabinoids modulate dopamine release in the brain. Using fast cyclic voltammetry, single pulse stimulation (0.1 ms; 10 V) was applied every 5 min and peak dopamine release was measured with a carbon fibre microelectrode. Application of the D₂ receptor agonist, quinpirole, inhibited single pulse dopamine overflow in a concentration‐dependent manner (IC₅₀: 3.25 × 10^?8 M). The CB₁ receptor agonist WIN55212‐2 (WIN; 1 μM) had no effect on single pulse dopamine release (93.9 ± 6.6% at 60 min, n = 5) but attenuated the inhibitory effect of quinpirole (30 nM; quinpirole 39.0 ± 4.2% vs. quinpirole + WIN, 48.2 ± 3.7%, n = 5, p < 0.05). This affect was antagonized by the CB₁ receptor anatgonist [N‐(Piperidin‐1‐yl)‐5‐(4‐iodophenyl)‐1‐(2,4‐dichlorophenyl)‐4‐methyl‐1H‐pyrazole‐3‐carboxamide] (AM‐251, 1 μM). Dopamine release evoked by four pulses delivered at 1 Hz (4P1Hz) and 10 pulses delivered at 5 Hz (10P5Hz) was significantly inhibited by WIN [72.3 ± 7.9% control (peak 4 to 1 ratio measurement) and 66.9 ± 3.8% control (area under the curve measurement), respectively, p < 0.05; n = 6 for both]. Prior perfusion of WIN significantly attenuated the effects of quinpirole on multiple pulse‐evoked dopamine release (4P1Hz: quinpirole, 28.4 ± 4.8% vs. WIN + quinpirole, 52.3 ± 1.2%; 10P5Hz: quinpirole, 29.5 ± 1.3% vs. WIN + quinpirole, 59.4 ±7.1%; p < 0.05 for both; n = 6). These effects were also antagonized by AM‐251 (1 μM). This is the first report demonstrating a functional, antagonistic interaction between CB₁ receptors and D₂ autoreceptors in regulating rat striatal dopamine release.