Effects of dietary restriction on insulin resistance in obese mice.

In many cases, development of insulin resistance has been linked to obesity and may contribute to mechanism of aging. The role of diet, irrespective of degree of obesity, in modulating insulin resistance and development of age degeneration disease remains uncertain. Lowered blood glucose levels are commonly associated with diet restriction (DR), which is an intervention shown to successfully retard aging and age associated disease. The effects of DR on blood glucose and insulin resistance were measured in yellow obese (A(vy)/A), lean black (a/a) mice and in another common inbred strain (B6C3F1) (at three different ages). The yellow obese mice become diabetic as a result of an insulin receptor defect which is not clearly understood. Insulin responses and radioinsulin binding were assayed in yellow obese and lean black mice fed either ad libitum (AL) or DR diets (YAL, BAL, YDR and YAL, respectively) at four different circadian intervals. The B6C3F1 controls were fed either AL (CAL) or DR (CDR) and measures were made at six circadian stages and three different ages. Within 23 days, DR produced a significant loss in body weight and a time-dependent 22-55% reduction in basal blood glucose levels in the yellow obese mice. Additionally, exogenous insulin produced circadian stage dependent (at the time of food intake) reductions in blood glucose in the YDR animals that were not present in YAL animals. (125)I-Insulin binding in liver was increased nearly 2-fold in YDR and BDR mice during the time of day that animals were active and eating. (125)I-Insulin binding was two-fold-higher in CDR mice at 4, 12 and >24 months of age. Binding decreased as a function of age in both the CAL and CDR animals. However, even in the >24 month group the CDR animals were found to have levels of binding that were as high as those found in younger CAL liver. The mechanism of action appears to be through resolution of insulin resistance by modulating an insulin receptor defect.

A circadian study of liver antioxidant enzyme systems of female Fischer-344 rats subjected to dietary restriction for six weeks.

We examined the influences of dietary restriction (DR) on the circadian profile of liver catalase (CAT), glutathione peroxidase (GPx), and interacting systems required for removal of H2O2 (support systems), in 18-week old female Fischer 344 rats fed 60% of their ad libitum (AL) diet for six weeks. Food was presented to the DR animals during the early light-span. Regardless of diet, enzyme levels were generally consistent with circadian patterns. In CR animals, maximum activities often occurred at the time of food presentation. CAT and GPx activities generally were significantly higher in DR animals than in AL animals at the time of feeding. When assessing glucose-6-phosphate dehydrogenase (G6PDH) activity using saturating substrate (NADP(+)) concentrations, higher activities were seen at all times of day in the AL animals; however, when activity was measured in the presence of lower (i.e., physiologic) NADP(+) concentrations, the reverse was true. In contrast, glutathione reductase (GR) activity was not influenced by DR. Cytosolic levels of NADPH peaked and were higher in DR than in AL rodents prior to feeding. NADH levels were not influenced by diet, but did manifest a significant circadian pattern with a maximum occurring toward the middle of the dark span. These data suggest that even at a young age and following only a relatively brief duration of DR, there exists an enhanced enzymatic capability in rats subjected to DR to remove free radicals generated as a consequence of normal oxidative metabolism. Further, these data support emerging trends suggesting metabolic regulation of antioxidant defense systems in response to free radical generation.

Effects of ischemia-hypoxia induced by interruption of uterine blood flow on fetal rat liver and brain enzyme activities and offspring behavior.

The effects of acute perinatal ischemia-hypoxia on fetal liver and brain energy metabolism, fetal brain total free fatty acid concentration and subsequent offspring behavior were investigated in rats. Ischemia-hypoxia was induced at term either by ligation of the uterine blood vessels or submersion of the entire uterine horn in warmed saline. Fetuses of the adjacent horn served as within-dam controls for all assessments and fetuses of dams which had not undergone the surgical stress served as independent controls for enzyme assays. Ischemia-hypoxia was associated with reduced activity of fatty acid synthase in the liver and brain. Total free fatty acid concentration significantly increased in the fetal hypoxic brain. Pups not used for enzyme analyses were cross-fostered for behavioral assessments. Relative to the enzymatic alterations, there were few behavioral alterations associated with ischemia-hypoxia. At postnatal day 30, rats made hypoxic by ligation of the uterine blood vessels had decreased caudate nucleus and brain stem weights relative to within-dam controls. At postnatal day 85, rats made hypoxic by submersion of the uterine horn had decreased olfactory bulb weight. The results of this study indicate an initial acute response to a brief period of ischemia-hypoxia at term pregnancy in the fetal rat brain and liver.

New steroidal antiinflammatory antedrugs: steroidal [16 alpha,17 alpha-d]-3'-carbethoxyisoxazolines.

Novel steroidal antiinflammatory antedrugs, 11 beta,20-dihydroxy-3,20-dioxo-3'-(ethoxycarbonyl)-isoxazolino[16,17 - d]pregna-1,4-diene (2a) and 9-fluoro-11 beta,20-dihydroxy-3,20-dioxo-3'-ethoxy- carbonylisoxazolino[16,17-d]pregna-1,4-diene (2b) were prepared in 97% yield via 1,3-dipolar cycloaddition of carbethoxyformonitrile (CEFNO) to 11 beta,21-dihydroxy-3,20-dioxopregna-1,4,-16-triene (1a) and 11 beta,21-dihydroxy-3,20-dioxo-9-fluoropregna-1,4,16-triene (1b), respectively, which were prepared via five steps from prednisolone and 9-fluoroprednisolone, respectively. The treatment of steroids 2a and 2b with acetic anhydride in pyridine led to the corresponding 21-acetates 3a and 3b, respectively, in 95% yield. Dose-response profiles of the croton oil-induced ear edema bioassay in rats were used to calculate the following ID50 values (nmol/ear resulting in a 50% reduction of edema): prednisolone (P), 540 nmol; 2b, 135 nmol; and 3b, 101 nmol. Inhibition of edema did not exceed 50% following application of either 2a or 3a. Relative potency calculations indicated that 2b was 4-fold and 3b 5.3-fold more potent than the parent compound P when applied topically. No significant adverse systemic effects were seen following treatments with 3b. These results suggest that C-9-fluorination, side-chain hydroxy group esterification, and [16 alpha,17 alpha-d]-3'-carbethoxyisoxazoline additions to the conventional steroid P improve topical antiinflammatory activity without concomitant increases in adverse systemic activity.

Acute toxicity of ganciclovir: effect of dietary restriction and chronobiology.

The effect of diet, age and time of dose delivery on the mortality of female B6C3F1 mice from ganciclovir sodium (DHPG) was determined for both single (SD; 400 mg DHPG/kg, ip) and multiple doses (MD; same dose ip for 10 additional days) of the drug. Young (7-10 months) and middle-aged (MA; 19-22 months) mice (B6C3F1), both fed ad lib. (AL) and calorie restricted (CR), were dosed at 0, 6, 12 and 18 hr after lights on (HALO; SD study) and at 12.00 hr (MD study). The SD study mortality rate was 38% (AL) and 1.7% (CR) (P < 0.00001). Mortality was 53% (AL, young; P < 0.00001), over 20% (AL, MA), over 1.8% (CR, MA; P = 0.00004) or more than 1.7% (CR, young; P = 0.00002). Effects were independent of lean body mass differences between AL and CR mice. In the SD study, comparing AL mice only, the greatest mortality was seen in young mice at 6 HALO, (73%; P = 0.0034) and lowest mortality in MA mice at 12 HALO (8%; P = 0.026), whereas in the MD study mortality was 63% AL and 33% CR (P = 0.015). By age, MD mortality was 80% (AL, young; P = 0.0035), 50% (CR, MA), 47% (AL, MA), and 15% (CR, young; P = 0.0013). CR protected both young and MA mice in SD and young mice in MD. Lowest mortality for AL was at 12 HALO. It is suggested that dosing at 12 HALO may protect by decreasing DHPG uptake during a period of minimal DNA synthesis in the affected organ(s). CR and timing of DHPG dose may obviate the necessity to discontinue DHPG because of toxicity in humans. The most significant finding of this study is the impact of diet on mortality.

Dimerization and activation of porcine pancreatic phospholipase A2 via substrate level acylation of lysine 56.

The porcine pancreatic phospholipase A2-catalyzed hydrolysis of the water-soluble chromogenic substrate 4-nitro-3-octanoyloxybenzoate shows an initial latency phase similar to the one observed in the hydrolysis of aggregated phospholipids by the same enzyme. We report here that during the latency phase the enzyme undergoes a slow, autocatalytic, substrate-level acylation whereby in a few of the catalytic events the scissile octanoyl group of the substrate, normally transferred to water, is transferred to the epsilon-amino group of lysine 56. The N epsilon 56-octanoylphospholipase shows a strong tendency to dimerize in solution and thus may be separated from the monomeric native enzyme by gel filtration. Octanoylation of Lys-56 activates the enzyme some 180-fold toward 4-nitro-3-octanoyloxybenzoate and more than 100-fold toward monolayers of 1,2-didecanoyl-sn-glycero-3-phosphocholine. Acylation also attends the enzymatic hydrolysis of 1,2-dipalmitoyl-sn-glycero-3-phosphocholine with the incorporation of 1 eq of palmitate. Kinetic analysis of the early phase of reaction with 4-nitro-3-octanoyloxybenzoate shows that in this initial step the rate of activation is first order with respect to enzyme and substrate. A much more rapid, autocatalytic activation occurs in the later phases of the reaction where the activation of the enzyme is catalyzed by the activated enzyme itself. These findings with porcine pancreatic phospholipase A2, together with those relative to a snake venom enzyme monomer (Cho, W., Tomasselli, A. G., Heinrikson, R. L., and Kézdy, F. J. (1988) J. Biol. Chem. 263, 11237-11241), strongly support the proposal that interfacial activation of monomeric phospholipases is due to substrate-level autoacylation resulting in fully potentiated dimeric enzymes.

Effect of morphine on the rat gastrointestinal cholinergic enzymes.

Male Sprague-Dawley rats weighing approximately 225 g and maintained under controlled lighting and temperature conditions, were used in this experiment. Animals were given morphine (10 mg/kg, i.p.) either at 10:00 hr or at 22:00 hr. One hour later, treated animals with their proper control were sacrificed and the mucosa of the stomach, duodenum, ileum and colon were separated and assayed for acetylcholinesterase (AChE) and choline acetyltransferase (ChAT) activities. Data obtained indicate that morphine administration during the light phase resulted in a significant increase (p less than 0.01) in AChE activity in the colon, with no significant effect on the stomach, duodenum or the ileum. There was also significant increase (p less than 0.05) in ChAT activity in the stomach and the duodenum as a result of morphine administration during the light phase, with no effect on other tissues studied. During the dark phase, there were no significant changes observed in AChE activity of all tissues examined following morphine administration. However, the determination of ChAT activity during the dark phase, following morphine treatment, showed a significant increase (p less than 0.01) in the duodenum and the ileum, with a significant decline (P less than 0.05) in the colon. Results obtained from this study indicate that gastrointestinal changes seen after morphine administration could be due to changes in the activity of the cholinergic enzymes and these changes may be diurnally controlled.

Brain cholinergic involvement in the diurnal variations of the rapid development of tolerance to the hypothermic effect of apomorphine.

Male Sprague-Dawley rats maintained under controlled environmental conditions were used. Choline acetyltransferase (ChAT) and acetylcholinesterase (AChE) activities were determined in the cerebral cortex, bulbus olfactorius, midbrain, hypothalamus, hippocampus, cerebellum, pons and medulla oblongata of control rats and rats treated with apomorphine (15 mg/kg, i.p.) after a single dose or after a second dose administered 24 h later at 10.00 or 22.00 h. Results of this experiment indicate that the repeated administration of apomorphine at 10.00 h was associated with rapid development of tolerance to the hypothermic action of this drug. A single injection of apomorphine resulted in significant (p less than 0.01) decrease of AChE and significant increase of ChAT activity (p less than 0.01) in the cortex, hypothalamus and pons. There were no significant differences between tolerant and control animals in the activities of AChE or ChAT in the bulbus olfactorius, cerebral cortex, midbrain, pons or medulla. On the other hand, repeated administration of apomorphine at 22.00 h was not associated with tolerance to the hypothermic action of this drug. A single injection of apomorphine at 22.00 h resulted in significant (p less than 0.01) increase in AChE activity of the midbrain, hippocampus, and the medulla oblongata with no significant changes in the cerebral cortex and bulbus olfactorius. There was a significant decline (p less than 0.01) of ChAT activity of the hypothalamus, hippocampus, cerebral cortex and the medulla oblongata (p less than 0.05). Meanwhile, there was a significant (p less than 0.05) increase of ChAT activity of the midbrain with no significant changes in the cerebellum, pons and the bulbus olfactorius.(ABSTRACT TRUNCATED AT 250 WORDS)

Brain cholinergic involvement during the rapid development of tolerance to morphine.

In this study, male Sprague-Dawley rats maintained under controlled environmental conditions were used. Choline acetyltransferase (ChAT) and acetylcholinesterase (AChE) activities were determined in cerebral cortex, bulbus olfactorius, midbrain, hypothalamus, hippocampus, cerebellum, pons and medulla oblongata in control rats and rats treated with morphine (10 mg/kg) for 1 or 2 days. Repeated administration of morphine was associated with a decline in the degree of analgesia produced. Significant increase (p less than 0.01) in AChE activity of the medulla oblongata was observed following morphine administration for 1 or 2 days. A single injection of morphine resulted in a significant decline (p less than 0.01) in ChAT activity of hypothalamus, cerebellum and medulla oblongata. However, no such decline could be observed after 2 consecutive daily injections of morphine. In the cerebral cortex there was a significant decline (p less than 0.01) in ChAT activity after the second administration of morphine. These findings indicate that the changes in the responsiveness of the brain cholinergic enzymes following repeated morphine administration may in part explain the rapid development of tolerance to the analgesic effect of morphine.

Effect of ethanol on the rat gastrointestinal cholinergic enzymes.

Male Sprague-Dawley rats weighing between 180 and 220 g and maintained under controlled lighting and temperature conditions were used in this experiment. Animals were given ethanol (3 g/kg, p.o.) 24 h after fasting. One group was given ethanol at 10.00 h (light phase) and the other at 22.00 h (dark phase). One hour later, the treated animals with their proper controls were sacrificed and the mucosa of the stomach, duodenum, ileum and colon were separated and assayed for choline acetyltransferase (ChAT) and acetylcholinesterase (AChE) activities. Data obtained indicate that the administration of ethanol resulted in significant decline (p less than 0.01) in ChAT activity in the stomach and the colon during the light phase. A significant increase (p less than 0.01) in ChAT activity was also noted in the ileum during the dark phase. There was a significant decrease (p less than 0.01) in AChE activity in the stomach during the dark phase. The administration of ethanol also resulted in a significant decline in AChE activity (p less than 0.05) in the duodenum and the colon (p less than 0.01). The results obtained indicate that the gastrointestinal changes caused by ethanol administration may be related to changes in the cholinergic enzymes of the mucosa of the gastrointestinal tract.

Effect of stress and glucocorticoids on the gastrointestinal cholinergic enzymes.

Male Sprague-Dawley rats maintained under controlled lighting and temperature conditions were used in this experiment. Animals were exposed to acute cold (4 degrees C) and immobilization stress for one hour, exposed to cold stress for 7 days (chronic stress) or treated with corticosterone (2 mg/kg) 1 hr prior to sacrificing. Animals with their proper controls were sacrificed and the stomach, duodenum, ileum and colon were separated and assayed for choline acetyltransferase (ChAT) and acetylcholinesterase (AChE) activities. The data obtained indicated that exposure to acute stress resulted in significant decline in ChAT activity in all tissues studied. The administration of corticosterone resulted in significant decline in ChAT activity in all tissues studied except for the duodenum. Meanwhile, the exposure to chronic stress did not have any significant effect on ChAT activity. On the other hand, acute stress caused significant increase in AChE activity in all tissues studied except for the ileum and stomach. The duodenal AChE activity of stressed animals increased thirty-fold when compared to control. The administration of glucocorticoids significantly reduced AChE in all tissues studied, except for the duodenum and stomach where there was thirty-two-fold increase as compared to the control levels. The exposure to chronic stress also caused significant increase in AChE of all tissues studied, except for the colon. The results of this experiments indicate that the duodenal AChE is extremely sensitive to stress or glucocorticoids and that stress induced changes in the cholinergic enzymes of the gastrointestinal tract may be mediated by adrenal steroids.