Tissue-specific dysregulation of hexose-6-phosphate dehydrogenase and glucose-6-phosphate transporter production in db/db mice as a model of type 2 diabetes

Aims/hypothesis  

Tissue-specific amplification of glucocorticoid action through 11β-hydroxysteroid dehydrogenase type 1 (11β-HSD1) affects the development of the metabolic syndrome. Hexose-6-phosphate dehydrogenase (H6PDH) mediates intracellular NADPH availability for 11β-HSD1 and depends on the glucose-6-phosphate transporter (G6PT). Little is known about the tissue-specific alterations of H6PDH and G6PT and their contributions to local glucocorticoid action in db/db mice.     

Differential involvement of enkephalins in analgesic tolerance, locomotor sensitization, and conditioned place preference induced by morphine

In this study, the authors investigated the role of enkephalins in morphine-induced conditioned place preference, locomotor sensitization, and analgesic tolerance. Both preproenkephalin wild type (ppENK [+/+]) and knockout (ppENK [-/-]) mice showed similar preference for the morphine-paired chamber over the vehicle-paired chamber, indicating morphine induced comparable conditioned place preference in ppENK (+/+) and ppENK (-/-) mice. Sensitization developed to the motor stimulatory action of morphine after its repeated administration, but the magnitude of this response was not altered in ppENK (-/-) mice. However, as shown previously, ppENK (-/-) mice displayed blunted morphine analgesic tolerance. Taken together, the results suggest that enkephalins may be important for the development of analgesic tolerance but not for conditioned place preference or behavioral sensitization induced by morphine.     

Differential regulation of prohormone convertase 1/3, prohormone convertase 2 and phosphorylated cyclic-AMP-response element binding protein by short-term and long-term morphine treatment: implications for understanding the "switch" to opiate addiction

Drug addiction is a state of altered brain reward and self-regulation mediated by both neurotransmitter and hormonal systems. Although an organism's internal system attempts to maintain homeostasis when challenged by exogenous opiates and other drugs of abuse, it eventually fails, resulting in the transition from drug use to drug abuse. We propose that the attempted maintenance of hormonal homeostasis is achieved, in part, through alterations in levels of processing enzymes that control the ratio of active hormone to pro-hormone. Two pro-hormone convertases, PC1/3 and PC2 are believed to be responsible for the activation of many neurohormones and expression of these enzymes is dependent on the presence of a cyclic-AMP response element (CRE) in their promoters. Therefore, we studied the effects of short-term (24-h) and long-term (7-day) morphine treatment on the expression of hypothalamic PC1/3 and PC2 and levels of phosphorylated cyclic-AMP-response element binding protein (P-CREB). While short-term morphine exposure down-regulated, long-term morphine exposure up-regulated P-CREB, PC1/3 and PC2 protein levels in the rat hypothalamus as determined by Western blot analysis. Quantitative immunofluorescence studies confirmed these regulatory actions of morphine in the paraventricular and dorsomedial nucleus of the hypothalamus. Specific radioimmunoassays demonstrated that the increase in PC1/3 and PC2 levels following long-term morphine led to increased TRH biosynthesis as evidence by increased TRH/5.4 kDa C-terminal proTRH-derived peptide ratios in the median eminence. Promoter activity experiments in rat somatomammotrope GH3 cells containing the mu-opioid receptor demonstrated that the CRE(s) in the promoter of PC1/3 and PC2 is required for morphine-induced regulation of PC1/3 and PC2. Our data suggest that the regulation of the prohormone processing system by morphine may lead to alterations in the levels of multiple bioactive hormones and may be a compensatory mechanism whereby the organism tries to restore its homeostatic hormonal milieu. The down-regulation of PC1/3, PC2 and P-CREB by short-term morphine and up-regulation by long-term morphine treatment may be a signal mediating the switch from drug use to drug abuse.     

The endogenous OFQ/N/ORL-1 receptor system regulates the rewarding effects of acute cocaine

Previous studies have shown that orphanin FQ/nociceptin (OFQ/N), the endogenous ligand of the opioid receptor-like (ORL-1) receptor, reduces the rewarding and addictive properties of cocaine and other drugs of abuse. In the present study, using the conditioned place preference (CPP) paradigm, as an animal model of drug reward, we assessed whether the rewarding action of acute cocaine would be altered in mice lacking the ORL-1 receptor or in wild type mice treated with J-113397, an ORL-1 receptor antagonist, relative to their saline-treated controls. On day 1, mice were tested for their baseline place preferences, in which each mouse was placed in the neutral chamber of a three-chambered CPP apparatus, allowed to freely explore all the chambers and the amount of time that a mouse spent in each conditioning chamber was recorded for 15 min. On days 2-3, mice received once daily alternate-day saline/cocaine (15 or 30 mg/kg) conditioning for 30 min. On day 4, mice were tested for their postconditioning preferences, as described for day 1. In a subsequent study, the effect of J-113397 (3 mg/kg) on the rewarding action of acute cocaine (15 mg/kg) was also examined in wild type mice. Our results showed that mice lacking the ORL-1 receptor expressed greater CPP than their wild type littermates. Furthermore, the rewarding action of cocaine was enhanced in the presence of J-113397 in wild type mice. Together, the present results suggest that the endogenous OFQ/N/ORL-1 receptor system is involved in the rewarding action of acute cocaine.     

X-ray- and neutron-scattering studies of alpha-crystallin and evidence that the target protein sits in the fenestrations of the alpha-crystallin shell

PURPOSE: Alpha-crystallin, a ubiquitous molecular chaperone, is found in high concentrations in the lens. Its structure and precise mechanism of action, however, are unknown. The purpose of these experiments was to further the understanding of the chaperone function of alpha-crystallin. METHODS: X-ray- and neutron-solution-scattering studies were used to measure the radius of gyration of bovine lens alpha-crystallin when complexed with its target protein beta-crystallin in both normal and heavy-water-based solutions. Spectrophotometry was used as a chaperone assay. RESULTS: The radius of gyration of alpha-crystallin on its own and when mixed with beta-crystallin was 69 +/- 1 A at 35 degrees C and increased with the temperature. In contrast to H2O-buffered solutions, the radius of gyration did not increase significantly in D2O-buffered solutions up to 55 degrees C, and at 70 degrees C was, on average, some 15 to 20 A smaller. CONCLUSIONS: Bovine lens alpha-crystallin in solution can be modeled as a fenestrated spherical shell of diameter 169 A. At physiological temperatures, a weak interaction between alpha- and beta-crystallin occurs, and beta-crystallin is located in the fenestrations. Deuterium substitution indicates that the superaggregation process is controlled by hydrogen bonding. However, the chaperone process and superaggregation appear not to be linked.     

The role of opioid receptor density in morphine tolerance.

The effect of chronic opioid antagonist-induced functional supersensitivity and receptor upregulation on morphine tolerance was examined in Swiss Webster mice obtained from Taconic Farms and Charles River Laboratories. Mice were implanted s.c. with either naltrexone (NTX) or placebo pellets for 8 days. After 8 days, the pellets were removed, and 24 h later mice were either injected with morphine (50 or 100 mg/kg) or saline (acute tolerance protocol) or implanted with a 75-mg morphine or placebo pellet for 72 h (chronic tolerance protocol). Mice were tested for morphine analgesia 24 h after injections or 72 h after pellet implantations. Mice from Taconic Farms were more sensitive to morphine analgesia than Charles River mice, although the degree of tolerance in both strains was similar. Acute morphine produced a 1.7- and 1.9-fold increase in the ED50 for morphine analgesia in NTX and placebo pretreated mice, respectively. the chronic tolerance protocol produced the same shift (2.4-fold) in the ED50 in both NTX and placebo pretreated mice. In both tolerance protocols, NTX-pretreated mice were significantly more sensitive to the analgesic effects of morphine than placebo pretreated controls. Binding studies ([3H][D-Ala,2NMePhe4,Gly-ol5]enkephalin) indicated an approximately 40% increase in opioid receptor density with no significant alteration in affinity after chronic NTX treatment. These results indicate that acute and chronic tolerance to morphine develops comparably in control and upregulated, supersensitive mice. These findings suggest that new binding sites in upregulated mice mediate tolerance similarly to existing binding sites and that the degree of tolerance is unrelated to the density of opioid receptors.     

Tolerance develops to spinal morphine analgesia but not morphine-induced convulsions

Administration of morphine into the spinal intrathecal (i.t.) space produced dose-dependent analgesia in the mouse. At higher doses i.t. morphine induced seizures of the hindlimbs. Mice treated chronically with morphine (75 mg pellet, s.c.) for 72 h were tolerant to the analgesic effects of i.t. morphine, but not to the proconvulsant action. Spinal morphine analgesia was attenuated by naloxone, whereas i.t. morphine-induced seizures were not. These results indicate that spinal opioid receptors mediate analgesia but not seizures following i.t. morphine treatment in the mouse.     

Dissociation of opioid receptor upregulation and functional supersensitivity.

Alterations in brain opioid binding and opioid pharmacodynamics following chronic (8-day) naltrexone (NTX) treatment were determined in pertussis toxin (PTX)-treated mice. Intrathecal (IT) and intracerebroventricular (ICV) PTX produced a time-dependent, long-lasting inhibition of morphine (SC) analgesia without modifying basal nociception. Inhibition was maximal 16 days following PTX treatment, and was still observed at 40 days. Relative to placebo controls, NTX treatment produced supersensitivity to morphine analgesia in all control mice and in mice pretreated with PTX 1 day before NTX. Supersensitivity was not observed in 7-day PTX-pretreated mice. [3H][D-Ala2-D-Leu5]enkephalin ([3H]DADLE) and [3H][D-Ala2-MePhe4-Gly(ol)5]enkephalin ([3H]DAMGO) binding sites were increased by NTX treatment in saline- and PTX-pretreated groups. KDs were unchanged. These results indicate that PTX does not alter opioid antagonist-induced receptor upregulation. However, PTX treatment can diminish morphine potency in upregulated and control mice. Therefore, opioid analgesia in control and upregulated mice appears to be mediated by receptors linked to a common PTX-sensitive G-protein. Furthermore, in 7-day PTX-pretreated mice, NTX increased binding sites without altering morphine potency, which suggests that new binding sites can appear without being functionally coupled.