Epac2a-null mice exhibit obesity-prone nature more susceptible to leptin resistance.

BACKGROUND: The exchange protein directly activated by cAMP (Epac), which is primarily involved in cAMP signaling, has been known to be essential for controlling body energy metabolism. Epac has two isoforms: Epac1 and Epac2. The function of Epac1 on obesity was unveiled using Epac1 knockout (KO) mice. However, the role of Epac2 in obesity remains unclear. METHODS: To evaluate the role of Epac2 in obesity, we used Epac2a KO mice, which is dominantly expressed in neurons and endocrine tissues. Physiological factors related to obesity were analyzed: body weight, fat mass, food intake, plasma leptin and adiponectin levels, energy expenditure, glucose tolerance, and insulin and leptin resistance. To determine the mechanism of Epac2a, mice received exogenous leptin and then hypothalamic leptin signaling was analyzed. RESULTS: Epac2a KO mice appeared to have normal glucose tolerance and insulin sensitivity until 12 weeks of age, but an early onset increase of plasma leptin levels and decrease of plasma adiponectin levels compared with wild-type mice. Acute leptin injection revealed impaired hypothalamic leptin signaling in KO mice. Consistently, KO mice fed a high-fat diet (HFD) were significantly obese, presenting greater food intake and lower energy expenditure. HFD-fed KO mice were also characterized by greater impairment of hypothalamic leptin signaling and by weaker leptin-induced decrease in food consumption compared with HFD-fed wild-type mice. In wild-type mice, acute exogenous leptin injection or chronic HFD feeding tended to induce hypothalamic Epac2a expression. CONCLUSIONS: Considering that HFD is an inducer of hypothalamic leptin resistance and that Epac2a functions in pancreatic beta cells during demands of greater work load, hypothalamic Epac2a may have a role in facilitating leptin signaling, at least in response to higher metabolic demands. Thus, our data indicate that Epac2a is critical for preventing obesity and thus Epac2a activators may be used to manage obesity and obesity-mediated metabolic disorders.

Association of thymosin ß4 expression with clinicopathological parameters and clinical outcomes of bladder cancer patients.

The clinical significance of thymosin ß4 (Tß4) expression in bladder transitional cell carcinoma (BTCC) remains unclear. The present study assessed the relationship between the expression of Tß4 protein and the clinicopathological features, as well as the prognosis of bladder cancer patients. Tß4 protein expression in 24 normal bladder and 138 primary BTCC tissue specimens was detected by immunohistochemistry, and the association of this expression with BTCC clinicopathological features and recurrence as well as patient survival was analyzed. Tß4 expression was significantly stronger in BTCC patients than in normal volunteers. The expression of Tß4 was significantly associated with differentiation capability, tumor stage and lymph node metastasis (P = 0.025, 0.043, and 0.039, respectively). Moreover, Tß4 expression was positively correlated with integrin-linked kinase (ILK) and ß-catenin expression (P = 0.042, 0.031, respectively) and inversely correlated with E-cadherin expression (P = 0.022). In the present cohort of bladder cancer patients, Tß4 expression was found to be a predictor of poor survival (P < 0.05); however, high Tß4 expression exhibited unfavorable prognostic value for recurrence. These data suggested that Tß4 is correlated with the pathogenesis of BTCC. In addition, the patients with higher Tß4 expression had a shorter survival.

Clinical significance of enzymatic lysophosphatidylcholine (LPC) assay data in patients with sepsis.

Lysophosphatidylcholine (LPC) has been suggested to serve as a useful prognostic marker for sepsis. However, existing LPC assays are complicated, time-consuming, and of limited application in real clinical situations. Thus, we investigated the serum LPC levels in sepsis patients using an enzymatic assay and analyzed the correlations between the serum LPC concentration and clinical characteristics. We prospectively collected blood samples from suspected sepsis patients, commencing on day 1 of sepsis. We analyzed all samples using an enzymatic assay. Additionally, we analyzed the serum LPC concentrations in a control group of 21 healthy blood donors. A total of 105 patients who fulfilled the sepsis criteria were included. The mean serum LPC concentration was 43.49 ± 33.09 µmol/L in sepsis patients, which was much lower than that of 21 healthy controls (234.68 ± 30.33 µmol/L, p<0.001). Bacteremic sepsis was associated with a lower serum LPC concentration than non-bacteremic sepsis (34.8 ± 26.85 vs. 49.05 ± 35.63 µmol/L, p<0.05). No difference in serum LPC concentration was evident between survivors and non-survivors. The serum LPC concentration tended to decrease with the severity of sepsis. The day 1 serum LPC concentration was decreased in patients with sepsis, especially when bacteremia was present. However, the serum LPC level did not correlate with disease severity and did not predict mortality from sepsis.

Cyclin-dependent kinase-5 prevents neuronal apoptosis through ERK-mediated upregulation of Bcl-2.

Cyclin-dependent kinase-5 (Cdk5) is required for neuronal survival, but its targets in the apoptotic pathways remain unknown. Here, we show that Cdk5 kinase activity prevents neuronal apoptosis through the upregulation of Bcl-2. Treatment of SH-SY5Y cells with retinoid acid (RA) and brain-derived neurotrophic factor (BDNF) generates differentiated neuron-like cells. DNA damage triggers apoptosis in the undifferentiated cells through mitochondrial pathway; however, RA/BDNF treatment results in Bcl-2 upregulation and inhibition of the mitochondrial pathway in the differentiated cells. RA/BDNF treatment activates Cdk5-mediated PI3K/Akt and ERK pathways. Inhibition of Cdk5 inhibits PI3K/Akt and ERK phosphorylation and Bcl-2 expression, and thus sensitizes the differentiated cells to DNA-damage. Inhibition of ERK, but not PI3K/Akt, abrogates Cdk5-medidated Bcl-2 upregulation and the protection of the differentiated cells. This study suggests that ERK-mediated Bcl-2 upregulation contributes to BDNF-induced Cdk5-mediated neuronal survival.

Mechanical properties for irradiated face-centred cubic nanocrystalline metals.

In this paper, a self-consistent plasticity theory is proposed to model the mechanical behaviours of irradiated face-centred cubic nanocrystalline metals. At the grain level, a tensorial crystal model with both irradiation and grain size effects is applied for the grain interior (GI), whereas both grain boundary (GB) sliding with irradiation effect and GB diffusion are considered in modelling the behaviours of GBs. The elastic-viscoplastic self-consistent method with considering grain size distribution is developed to transit the microscopic behaviour of individual grains to the macroscopic properties of nanocrystals (NCs). The proposed theory is applied to model the mechanical properties of irradiated NC copper, and the feasibility and efficiency have been validated by comparing with experimental data. Numerical results show that: (i) irradiation-induced defects can lead to irradiation hardening in the GIs, but the hardening effect decreases with the grain size due to the increasing absorption of defects by GBs. Meanwhile, the absorbed defects would make the GBs softer than the unirradiated case. (ii) There exists a critical grain size for irradiated NC metals, which separates the grain size into the irradiation hardening dominant region (above the critical size) and irradiation softening dominant region (below the critical size). (iii) The distribution of grain size has a significant influence on the mechanical behaviours of both irradiated and unirradiated NCs. The proposed model can offer a valid theoretical foundation to study the irradiation effect on NC materials.

Glimepiride block of cloned beta-cell, cardiac and smooth muscle K(ATP) channels.

1. We examined the effect of the sulphonylurea glimepiride on three types of recombinant ATP-sensitive potassium (K(ATP)) channels. 2. K(ATP) channels share a common pore-forming subunit, Kir6.2, which associates with different sulphonylurea receptor isoforms (SUR1 in beta-cells, SUR2A in heart and SUR2B in smooth muscle). 3. Kir6.2 was coexpressed with SUR1, SUR2A or SUR2B in Xenopus oocytes and macroscopic K(ATP) currents were recorded from giant inside-out membrane patches. Glimepiride was added to the intracellular membrane surface. 4. Glimepiride inhibited Kir6.2/SUR currents by interaction with two sites: a low-affinity site on Kir6.2 (IC(50)= approximately 400 microM) and a high-affinity site on SUR (IC(50)=3.0 nM for SUR1, 5.4 nM for SUR2A and 7.3 nM for SUR2B). The potency of glimepiride at the high-affinity site is close to that observed for glibenclamide (4 nM for SUR1, 27 nM for SUR2A), which has a similar structure. 5. Glimepiride inhibition of Kir6.2/SUR2A and Kir6.2/SUR2B currents, but not Kir6.2/SUR1 currents, reversed rapidly. 6. Our results indicate that glimepiride is a high-affinity sulphonylurea that does not select between the beta-cell, cardiac and smooth muscle types of recombinant K(ATP) channel, when measured in inside-out patches. High-affinity inhibition is mediated by interaction of the drug with the sulphonylurea receptor subunit of the channel.

Central injection of nitric oxide synthase inhibitors increases peripheral interleukin-6 and serum amyloid A: involvement of adrenaline from adrenal medulla.

1. Accumulating evidence suggests that plasma levels of interleukin-6 (IL-6), a major cytokine stimulating the synthesis of acute phase proteins, are intimately regulated by the central nervous system (CNS). 2. In the present study, effects of intracerebroventricular (i.c. v) injection of N(G)-nitro-L-arginine methyl ester (L-NAME) or 7-nitroindazole, nitric oxide synthase (NOS) inhibitors, on plasma IL-6 levels and peripheral IL-6 mRNA expression were examined in mice. 3. L-NAME (0.1 - 2 microg per mouse i.c.v.) and 7-nitroindazole (0.2 - 2 microg per mouse i.c.v.) induced a dose-dependent increase in plasma IL-6 levels and a subsequent increase in circulating serum amyloid A, a liver acute-phase protein. In contrast, an intraperitoneal (i.p.) injection of L-NAME up to the dose of 25 microg per mouse had no effect. 4. Pretreatment with yohimbine (alpha(2)-adrenergic antagonist; 1 mg kg(-1) i.p.), or ICI-118,551 (beta(2)-adrenergic antagonist; 2 mg kg(-1) i.p.), but not with prazosin (alpha(1)-adrenergic antagonist; 1 mg kg(-1) i.p.), nor betaxolol (beta(1)-adrenergic antagonist; 2 mg kg(-1) i.p.), significantly inhibited the central L-NAME-induced plasma IL-6 levels. 5. I.c.v. (50 microg per mouse) or i.p. (100 mg kg(-1)) pretreatment with 6-hydroxydopamine had no effect on central L-NAME-induced plasma IL-6 levels. However, intrathecal (i.t.) pretreatment with 6-hydroxydopamine (20 microg per mouse) markedly inhibited central L-NAME-induced plasma IL-6 levels. Both yohimbine (1.5 microg per mouse i.t.) and ICI-118,551 (1.5 microg per mouse i. t.) were effective in inhibition of central L-NAME-induced plasma IL-6 levels. 6. There was an elevation of base-line plasma IL-6 levels in adrenalectomized animals. The adrenalectomy-enhanced levels were not further increased by central L-NAME. 7. L-NAME (2 microg per mouse i.c.v.) induced an increase in IL-6 mRNA expression in liver, spleen, and lymph node. 8. These results suggest that NOS activity in the brain tonically down-regulates peripheral IL-6 by inhibiting adrenaline release from the adrenal medulla.

Protection against beta-amyloid peptide toxicity in vivo with long-term administration of ferulic acid.

1. beta-Amyloid peptide (A beta), a 39 -- 43 amino acid peptide, is believed to induce oxidative stress and inflammation in the brain, which are postulated to play important roles in the pathogenesis of Alzheimer's disease. Ferulic acid is an antioxidant and anti-inflammatory agent derived from plants; therefore, the potential protective activity of ferulic acid against A beta toxicity in vivo was examined. 2. Mice were allowed free access to drinking water (control) or water containing ferulic acid (0.006%). After 4 weeks, A beta 1-42 (410 pmol) was administered via intracerebroventricular injection. 3. Injection of control mice with A beta 1-42 impaired performance on the passive avoidance test (35% decrease in step-through latency), the Y-maze test (19% decrease in alternation behaviour), and the water maze test (32% decrease in percentage time in platform-quadrant). In contrast, mice treated with ferulic acid prior to A beta 1-42 administration were protected from these changes (9% decrease in step-through latency; no decrease in alternation behaviour; 14% decrease in percentage time in platform-quadrant). A beta 1-42 induced 31% decrease in acetylcholine level in the cortex, which was tended to be ameliorated by ferulic acid. 4. In addition, A beta 1-42 increased immunoreactivities of the astrocyte marker glial fibrillary acidic protein (GFAP) and interleukin-1 beta (IL-1 beta) in the hippocampus, effects also suppressed by pretreatment with ferulic acid. 5. Administration of ferulic acid per se unexpectedly induced a transient and slight increase in GFAP and IL-1 beta immunoreactivity in the hippocampus on day 14, which returned to basal levels on day 28. A slight (8%) decrease in alternation behaviour was observed on day 14. 6. These results demonstrate that long-term administration of ferulic acid induces resistance to A beta 1-42 toxicity in the brain, and suggest that ferulic acid may be a useful chemopreventive agent against Alzheimer's disease.

Overexpression of short heterodimer partner recovers impaired glucose-stimulated insulin secretion of pancreatic beta-cells overexpressing UCP2.

The short heterodimer partner (SHP) (NR0B2) is an orphan nuclear receptor whose function in pancreatic beta-cells is unclear. Mitochondrial uncoupling protein (UCP2) in beta-cells is upregulated in obesity-related diabetes, causing impaired glucose-stimulated insulin secretion (GSIS). We investigated whether SHP plays a role in UCP2-induced GSIS impairment. We overexpressed SHP in normal islet cells and in islet cells overexpressing UCP2 by an adenovirus-mediated infection technique. We found that SHP overexpression enhanced GSIS in normal islets, and restored GSIS in UCP2-overexpressing islets. SHP overexpression increased the glucose sensitivity of ATP-sensitive K+ (KATP) channels and enhanced the ATP/ADP ratio. A peroxisome proliferator-activated receptor gamma (PPARgamma) antagonist, GW9662, did not block the SHP effect on GSIS. SHP overexpression also corrected the impaired sensitivity of UCP2-overexpressing beta-cells to methylpyruvate, another energy fuel that bypasses glycolysis and directly enters the Krebs cycle. KATP channel inhibition mediated by dihydroxyacetone, which gives reducing equivalents directly to complex II of the electron transport system, was similar in Ad-Null-, Ad-UCP2- and Ad-UCP2+Ad-SHP-infected cells. The mitochondrial metabolic inhibitor sodium azide totally blocked the effect of SHP overexpression on GSIS. These results suggest that SHP positively regulates GSIS in beta-cells and restores glucose sensitivity in UCP2-overexpressing beta-cells by enhancing mitochondrial glucose metabolism, independent of PPARgamma activation.

The effect of cycloheximide on the regulation of proenkephalin and prodynorphin gene expressions induced by kainic acid in rat hippocampus.

The effect of cycloheximide (CHX), a protein synthesis inhibitor, on the regulation of proenkephalin (proENK) and prodynorphin (proDYN) mRNA levels, proto-oncogenes, such as c-fos, 35-kDa fra and c-jun mRNA, and the levels of their products induced by kainic acid (KA) in rat hippocampus was studied. The proENK and proDYN mRNA levels were markedly increased 4 and 8 h after KA (10 mg/kg i.p.) administration. However, the intracellular proENK protein level was not affected by KA. The elevations of both proENK and proDYN mRNA levels induced by KA were inhibited by pre-administration of CHX (15 mg/kg i.p.). The increases of proENK and proDYN mRNA levels induced by KA were well-correlated with the increases of c-Fos, 35-kDa Fra and c-Jun protein levels. KA administration increased the hippocampal levels of c-Fos, 35-kDa Fra and c-Jun proteins with the time. The increases of c-Fos, 35-kDa Fra and c-Jun protein levels induced by KA administration were also inhibited by CHX pre-administration. KA administration markedly increased both c-fos and c-jun mRNA levels during 1 and 4 h and the increased levels of these proto-oncogene mRNA were further prolonged by the treatment with CHX. In addition, CHX alone increased both c-fos and c-jun mRNA levels although the onset times of induction were different. In electrophoretic mobility shift-assay, both AP-1 and ENKCRE-2 DNA-binding activities were increased by KA. KA-induced increases of AP-1 and ENKCRE-2 DNA-binding activities were also attenuated by CHX. In addition, KA-induced AP-1 and ENKCRE-2 DNA-binding activities were diminished by the antibodies against Fos and Jun family proteins. Furthermore, the cross-competition studies revealed that AP-1 proteins actively participated in ENKCRE-2 DNA domain. The results suggest that KA-induced proENK and proDYN mRNA expressions may require on-going synthesis of proteins, such as c-Fos, c-Jun and 35-kDa Fra, which may have a possible role in the up-regulation of proENK and proDYN gene expression through the binding with AP-1 and ENKCRE-2 DNA-binding motifs.

The stimulation of rat astrocytes with phorbol-12-myristate-13-acetate increases the proenkephalin mRNA: involvement of proto-oncogenes.

The effect of phorbol-12-myristate-13-acetate (PMA) on the regulation of proenkephalin (proENK) mRNA level, ENKCRE-2 or AP-1 DNA binding activity, and the mRNA and protein levels of proto-oncogenes (c-fos, fra-1, and c-jun) in primary cultured rat astrocytes were studied. The proENK mRNA level was elevated at 4 h after the treatment of PMA (2.5 microM) without altering the intracellular proENK protein level, and this increase was attenuated by pre-treatment with cycloheximide (CHX; 15 microM), a protein synthesis inhibitor. Both AP-1 and ENKCRE-2 DNA binding activities were markedly increased at 1-4 h by PMA treatment and these PMA-induced responses were inhibited by pre-treatment with CHX, showing that the increase of proENK mRNA level was well correlated with the AP-1 and ENKCRE-2 DNA binding activities. In contrast, although the phospho-CREBP level was also increased by PMA at 0.5-1 h, the pre-treatment with CHX further increased the PMA-induced phospho-CREBP level. In addition, PMA caused the induction of c-fos, c-jun and fra-1 mRNA level and, especially, PMA-induced increase of fra-1 mRNA level was further enhanced by CHX treatment at 4 h. Furthermore, western immunoblot assay showed that PMA caused induction of c-Fos, Fra-1, and c-Jun protein levels. PMA-induced increases of proto-oncoproteins levels were also inhibited by CHX treatment. The results suggest that newly synthesized AP-1 proteins, such as c-Fos, Fra-1, and c-Jun may play important roles in the regulation of PMA-induced proENK gene expression in cultured rat astrocytes. Phospho-CREB protein appears not to be involved in the regulation of PMA-induced proENK gene expression.

Stimulation of astrocyte-enriched culture with arachidonic acid increases proenkephalin mRNA: involvement of proto-oncoprotein and mitogen activated protein kinases.

In astrocyte-enriched cultures, arachidonic acid (AA, 100 microM) significantly increased the proenkephalin (proENK) mRNA level (4. 9-fold at 8 h). In addition, AA also increased several AP-1 proteins, such as c-Fos, Fra-1, Fra-2, JunB, JunD, and c-Jun, or AP-1 and ENKCRE-2 DNA-binding activity. As well as AP-1 proteins and their DNA-binding activities, proENK mRNA level induced by AA was reduced by the pretreatment with 15 microM of cycloheximide (CHX; 1.6-fold). AA-dependent increase of proENK mRNA is not mediated by cyclooxygenase- or lipoxygenase-dependent metabolites, or free radicals, because the AA-induced increase of proENK mRNA levels was not affected by indomethacin (10 microM), nordihydroguaiaretic acid (10 microM), or N-acetylcysteine. However, as well as proto-oncoprotein levels, such as Fra-1, Fra-2, c-Jun, JunB, but not JunD, AA-induced increase of proENK mRNA was significantly reduced by the pretreatment with 10 microM of PD98059 (1.3-fold) or 10 microM of SB203580 (1.8-fold). These results strongly suggest that AA rather than one of its metabolites is involved in the increase of proENK mRNA. In addition, the activation of both the p38 and ERK pathways appears to be involved in the AA-induced increase of proENK mRNA via activating the expression of proto-oncoprotein, such as Fra-1, Fra-2, c-Jun, and JunB.

The modulatory role of nitric oxide in the regulation of proenkephalin and prodynorphin gene expressions induced by kainic acid in rat hippocampus.

The effect of L-arginine (L-ARG), a nitric oxide donor, or Nomega-nitro-L-arginine (L-NAME), a nitric oxide synthase inhibitor, on the regulation of kainic acid (KA)-induced proenkephalin (proENK) and prodynorphin (proDYN) mRNA expressions in rat hippocampus was studied. The proENK and proDYN mRNA levels were markedly increased 6 h after KA (10 mg/kg, i.p.) administration. The elevations of both proENK and proDYN mRNA levels induced by KA was effectively inhibited by pre-administration of L-ARG (400 mg/kg, i.p.), but was not affected by pre-treatment with L-NAME (200 mg/kg, i.p.). The blockade of KA-induced proENK and proDYN mRNA levels by the pre-treatment with L-ARG was well correlated with proto-oncoprotein levels, such as c-Fos, Fra-2, FosB, JunD, JunB, and c-Jun, as well as AP-1 and ENKCRE-2 DNA binding activities. The pre-administration with L-NAME further increased KA-induced c-jun and c-fos mRNA levels in addition to their protein product levels, although the pre-treatment with L-NAME did not affect KA-induced FosB, Fra-2, JunB, and JunD protein levels at 6 h after treatment. In addition, the pre-administration with L-NAME further increased the KA-induced AP-1 and ENKCRE-2 DNA binding activities. Our results suggest that L-ARG plays an important role in inhibiting KA-induced proENK or proDYN mRNA expression, and its inhibitory action may be mediated through reducing the proto-oncoprotein levels, such as c-Fos, Fra-2, FosB, c-Jun, JunD, and JunB. In addition, L-NAME potentiated the c-Fos or c-Jun gene expression, as well as AP-1 or ENKCRE-2 DNA binding activity. However, these increases did not show the potentiative effect on KA-induced increases of proENK and proDYN mRNA level.

Prostaglandin E2 increases proenkephalin mRNA level in rat astrocyte-enriched culture.

The effect of prostaglandin E2 (PGE2) on proenkephalin (proENK) mRNA expression in primary cultured rat astrocytes was studied. The proENK mRNA level was significantly increased about 3.3-fold 4 h after PGE2 (10 microM) treatment and this increase was potentiated by the pre-treatment with cycloheximide (CHX; 15 microM) about 1.7-fold as much as PGE2 alone treated cells. The pretreatment with staurosporine (1 microM) completely inhibited the increase of PGE2-induced proENK mRNA level, although only a partial inhibition of PGE2-induced proENK mRNA level (approximately 1.5-fold) by H89 (10 microM) was observed. The increase of PGE2-induced proENK mRNA level was not affected by the pretreatment with PD98059 (1, 5, and 10 microM), omega-conotoxin GIVA (1 microM), nimodipine (1 microM), calmidazolium (1 microM), or KN-62 (1 microM). In addition to the proENK mRNA level, PGE2 also increased c-Fos (approximately 4.3-fold), Fra-1 ( approximately 3.8 fold), and Fra-2 (approximately 8.2-fold) protein levels at 4 h after drug treatment. However, c-Jun, JunB, and JunD protein levels were not affected by PGE2. Indeed, PGE2 failed to up-regulate c-jun mRNA expression as well as its protein product. Surprisingly, although three Jun proteins were not induced by PGE2, AP-1 and ENKCRE-2 DNA binding activities were increased by PGE2, (approximately 5 and approximately 2.8-fold, respectively) and which were effectively reduced by CHX (approximately 2.5 and 2-fold, respectively). In western blot analyses, PGE2 enhanced the phosphorylation of CREB (approximately 2.6-fold at 1 h), and CHX showed a potentiative effect on PGE2-induced CREB phosphorylation ( approximately 1.7 fold at 1 h) which is similar to the action on proENK mRNA regulation. Our results suggest that PGE2 increases proENK mRNA expression via activating serine/threonine protein kinase such as PKA, but not calcium/calmodulin dependent protein kinase and MAPK. In addition, phosphorylation of CREB rather than the increase of AP-1 may have a possible role at least early stage in PGE2-induced proENK mRNA level and CHX-evoked potentiation.

Evidence of enhancement of malate-aspartate shuttle activity in beta cells of streptozotocin-induced non-insulin-dependent diabetic rats.

Glucose-induced insulin secretion is selectively impaired in beta cells from animals with non-insulin-dependent diabetes mellitus (NIDDM). This study was performed to clarify whether the malate-aspartate shuttle among the glucose metabolic pathways is intact in beta cells of NIDDM rats. The insulin secretory capacity of the islets and the K(ATP) channel activity in single beta cells were measured in control and NIDDM rats injected with streptozotocin (STZ) during the neonatal period, using a radioimmunoassay and patch-clamp technique. The increase of insulin secretion induced by 11.1 mmol/L glucose or 10 mmol/L dihydroxyacetone (DHA) was significantly reduced in NIDDM islets, suggesting an impaired glycerol-phosphate shuttle. The application of glyceraldehyde (10 mmol/L) in NIDDM or control islets elicited an increase in insulin secretion, but the difference between the 2 groups was indistinguishable. On the contrary, the increase of insulin secretion and the inhibition of K(ATP) channel activity induced by aspartate, which preferentially participates in the malate-aspartate shuttle, were significantly greater in NIDDM versus the control. However, intracellularly applied aspartate in the inside-out mode did not inhibit K(ATP) channel activity. These findings show that malate-aspartate shuttle activity is potentiated in pancreatic beta cells of NIDDM rats, suggesting the development of a compensatory mechanism for the reduced activity of the glycerol-phosphate shuttle in NIDDM.

Inhibition of stress-induced plasma corticosterone levels by ginsenosides in mice: involvement of nitric oxide.

Ginseng total saponins (GTS) injected intracerebroventricularly (i.c.v.) at doses of 0.1-1 microg inhibited the i.c.v. injection stress-induced plasma corticosterone levels in mice. The inhibitory action of GTS was blocked by co-administered N(G)-nitro-L-arginine methyl ester (L-NAME; 1.5 microg, i.c.v.), an inhibitor of nitric oxide synthase (NOS). Of the ginsenosides Rb1, Rb2, Rc, Rd, Re, Rf, Rg1, 20(S)-Rg3 and 20(R)-Rg3 injected i.c.v. at doses of 0.01-1 microg, 20(S)-Rg3 and Rc significantly inhibited the i.c.v. injection stress-induced plasma corticosterone levels. The inhibitory actions of 20(S)-Rg3 and Rc were blocked by co-administered L-NAME (1.5 microg, i.c.v.). These results suggest that GTS, 20(S)-Rg3 and Rc may inhibit the i.c.v. injection stress-induced hypothalamo-pituitary-adrenal response by inducing NO production in the brain.

Involvement of different subtypes of cholecystokinin receptors in opioid antinociception in the mouse.

Various doses of sulfated cholecystokinin octapeptide (CCK-8s) injected intracerebroventricularly (ICV) alone did not show any antinociceptive effect. CCK-8s (0.01-1 ng) pretreated ICV for 10 min dose-dependently attenuated the inhibition of the tail flick response induced by ICV-administered morphine (2 micrograms). beta-endorphin (1 microgram), and U50,488H (trans-3,4-dichloro-N-methyl-N-[2-(1-pyrrolidinyl) cyclohexyl]benzeocetamide), 60 micrograms). However, ICV pretreatment with CCK-8s was not effective in reducing the inhibition of the tail flick response induced by [D-Pen(2)-D-Pen5]enkephalin (DPDPE; 10 micrograms) administered ICV. To determine what subtype(s) of CCK receptors are involved in antagonizing the antinociception induced by these opioids, effect of lorglumide sodium salt (a CCKA receptor antagonist) or PD135,158 N-methyl-D-glucamine salt (a CCKB receptor antagonist) on opioid-induced inhibition of the tail flick response was examined. Various doses of lorglumide sodium salt (lorglumide) or PD135,158 N-methyl-D-glucamine salt (PD135,158) injected ICV alone did not affect the basal tail flick response. The antagonistic effect of CCK-8s on morphine-, beta-endorphin-, and U50,488H-induced inhibition of the tail flick response was blocked in a dose-dependent manner by the co-ICV injection of PD135,158 (0.001-0.1 ng). The co-ICV injection of lorglumide (0.001-0.1 ng) dose-dependently blocked the antagonistic effect of CCK-8s on beta-endorphin- and U50,488H-induced, but not morphine-induced, inhibition of the tail flick response. Our results suggest that both CCKA and CCKB receptors are involved in antagonizing antinociception induced by beta-endorphin and U50,488H administered supraspinally. However, only CCKB (but not CCKA) receptors are involved in antagonizing antinociception induced by morphine administered supraspinally. CCK receptors are not involved in antagonizing the supraspinally administered DPDPE-induced antinociception.

Differential effects of adenosine receptor antagonists injected intrathecally on antinociception induced by morphine and beta-endorphin administered intracerebroventricularly in the mouse.

A previous study reported that beta-endorphin and morphine administered supraspinally produce antinociception by activating different descending pain inhibitory systems. The present study was designed to investigate the blocking effects of A1 or A2 adenosine receptors in the spinal cord on antinociception induced by supraspinally administered mu- and epsilon-opioid receptor agonists. The effects of 1,3-dipropyl-8-(2-amino-4-chloro-phenyl)-xanthine (PACPX; an A1 adenosine receptor antagonist) or 3,7-dimethyl-1-propargylxanthine (DMPX; an A2 adenosine receptor antagonist) on the antinociception induced by morphine (a mu-opioid receptor agonist) or beta-endorphin (an epsilon-opioid receptor agonist) administered intracerebroventricularly (i.c.v.) were studied. The antinociception was assayed by the tail-flick test. DMPX at doses of 1-40 micrograms (which administered intrathecally alone did not affect the latencies of tail-flick thresholds), attenuated dose-dependently the inhibition of the tail-flick response induced by i.c.v. administered morphine (0.5 microgram) or beta-endorphin (1 microgram). PACPX at doses of 1-40 micrograms (which administered intrathecally alone did not affect the latencies of tail-flick thresholds), attenuated dose-dependently the inhibition of the tail-flick response induced by i.c.v. administered beta-endorphin but not morphine. These results suggest that A2 but not A1 adenosine receptors in the spinal cord may be involved in the antinociception induced by supraspinally administered morphine, while the antinociception induced by supraspinally administered beta-endorphin appears to be mediated by spinal A1 and A2 adenosine receptors. These results support the hypothesis that morphine and beta-endorphin administered supraspinally produce antinociception by different neuronal mechanisms.

The effects of protection by D-Pen2-D-Pen5-enkephalin or D-Ala2-NMePhe4-Gly-ol-enkephalin against beta-chlornaltrexamine in the spinal cord on the antinociception induced by beta-endorphin administered intracerebroventricularly in the mouse.

Chlornaltrexamine (beta-CNA, 0.5 micrograms) alone or beta-CNA plus either mu-agonist, D-Ala2-NMePhe4-Gly-ol-enkephalin (DAMGO, 500 ng) or delta-agonist, D-Pen2-D-Pen5-enkephalin (DPDPE, 10 micrograms) was injected intrathecally (i.t.) to protect mu- or delta-opioid receptors, respectively, for 24 h in male ICR mice. The antinociception was assessed by the tail-flick and hot-plate test. DPDPE or DAMGO injected i.t. increased inhibition of the tail-flick and hot-plate response in a dose-dependent manner. The dose-response curve for tail-flick and hot-plate response induced by DPDPE or DAMGO in i.t. saline-treated group significantly shifted to the right in i.t. beta-CNA alone treated group but returned to the control level in the group treated with i.t. beta-CNA coadministered with DPDPE or DAMGO, respectively. The effects of protection of mu- and delta-opioid receptor in the spinal cord on inhibition of the tail-flick and hot-plate response induced by beta-endorphin and morphine administered intracerebroventricularly (i.c.v.) were then studied. Intrathecal pretreatment with beta-CNA or beta-CNA coadministered with DAMGO attenuated inhibition of the tail-flick response induced by beta-endorphin administered i.c.v. However, i.t. treatment with beta-CNA coadministered with DPDPE did not affect inhibition of the tail-flick response induced by beta-endorphin administered i.c.v. Intrathecal pretreatment with beta-CNA or beta-CNA coadministered with either DPDPE or DAMGO did not alter inhibition of the hot-plate response induced by beta-endorphin administered i.c.v.(ABSTRACT TRUNCATED AT 250 WORDS)

Differential effects of intrathecally injected galanin on antinociception induced by beta-endorphin and morphine administered intracerebroventricularly in mice.

The effects of intrathecal (i.t.) and intracerebroventricular (i.c.v.) treatments with galanin on inhibition of the tail-flick and paw-licking hot-plate responses induced by beta-endorphin and morphine administered i.c.v. were studied in ICR mice. Galanin (100 ng) given i.t. effectively antagonized inhibition of the tail-flick response induced by i.c.v. administered beta-endorphin (1 microgram) but not morphine (1 microgram). However, the same dose of galanin given i.t. did not affect inhibition of the hot-plate response induced by beta-endorphin and morphine administered i.c.v. Intrathecal treatment with various doses of galanin (0.1-100 ng) dose-dependently antagonized the inhibition of the tail-flick response induced by beta-endorphin administered i.c.v. Galanin (100 ng) in combination with beta-endorphin (1 microgram) or morphine (1 microgram) given i.c.v. did not affect beta-endorphin- or morphine-induced inhibition of the tail-flick and hot-plate responses. It is concluded that galanin given i.t. selectively attenuates i.c.v. beta-endorphin-induced inhibition of the tail-flick response by inhibiting descending epsilon-opioid system activated by supraspinally applied beta-endorphin.